ANTIBODY TARGETING OSTEOCLAST-RELATED PROTEIN Siglec-15

ABSTRACT

To provide a method of detecting abnormal bone metabolism by using a gene strongly expressed in an osteoclast; a method of screening a compound having a therapeutic and/or preventive effect on abnormal bone metabolism; and a pharmaceutical composition for treating and/or preventing abnormal bone metabolism. Provision of a method of detecting abnormal bone metabolism by using the expression of human Siglec-15 gene as an index; a pharmaceutical composition containing an antibody which specifically recognizes human Siglec-15 and has an activity of inhibiting osteoclast formation; and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/481,016, filed May 25, 2012, which is a Continuation of U.S.application Ser. No. 12/677,621, which is the U.S. National Stageapplication of PCT/JP2008/068287, filed Oct. 8, 2008, which claimspriority from Japanese Application No. JP 2007-265420, filed Oct. 11,2007, the entire contents of which are all incorporated herein byreference.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 12, 2015, isnamed sequence.txt and is 72 KB.

TECHNICAL FIELD

The present invention relates to a substance useful as a therapeuticand/or preventive agent for abnormal bone metabolism, a method ofscreening a substance useful as a therapeutic and/or preventive agentfor abnormal bone metabolism, a method of detecting abnormal bonemetabolism, and a method of treating and/or preventing abnormal bonemetabolism.

BACKGROUND

Bone is known to be a dynamic organ which is continuously remodeled byrepeated formation and resorption so as to change its own morphology andmaintain blood calcium levels. Healthy bone maintains an equilibriumbetween bone formation by osteoblasts and bone resorption byosteoclasts, and the bone mass is maintained constant. In contrast, whenthe equilibrium between bone formation and bone resorption is lost,abnormal bone metabolism such as osteoporosis occurs (EndocrinologicalReview, (1992) 13, pp. 66-80, Principles of Bone Biology, AcademicPress, New York, (1996) pp. 87-102,).

As factors which regulate bone metabolism, many systemic hormones andlocal cytokines have been reported, and these factors collaborate withone another to form and maintain bone (Endocrinological Review, (1992)13, pp. 66-80, Endocrinological Review, (1996) 17, pp. 308-332). As achange in bone tissue due to aging, the occurrence of osteoporosis iswidely known, but the mechanism of its occurrence encompasses variousfactors such as a decrease in secretion of sex hormones and anabnormality in the receptors for the hormones, variation in cytokineexpression locally in bone, expression of aging genes, and osteoclast orosteoblast differentiation failure or dysfunction, and thus, it isdifficult to consider it as a simple age-related physiologicalphenomenon. Primary osteoporosis is largely divided into postmenopausalosteoporosis due to a decrease in secretion of estrogen and senileosteoporosis due to aging, but advancement of basic research on themechanisms of regulation of bone formation and bone resorption isessential to elucidate the mechanism of its occurrence and to develop atherapeutic agent therefor.

Osteoclasts are multinucleated cells derived from hematopoietic stemcells, and by releasing chloride ions and hydrogen ions on a bonesurface to which osteoclasts adhere, osteoclasts acidify a gap betweenthe bone surface and the osteoclasts and also secrete cathepsin K whichis an acid protease or the like (American Journal of Physiology, (1991)260, C1315-C1324). This causes degradation of calcium phosphate,activation of acid proteases and degradation of bone matrix proteins,resulting in bone resorption.

Osteoclast precursor cells have been found to be differentiated intoosteoclasts by stimulation with RANKL (receptor activator of NF-κBligand) expressed on the cell membrane of osteoblasts/stromal cellspresent on the surface of bone (Proceedings of the National Academy ofScience of the United States of America, (1998) 95, pp. 3597-3602, Cell,(1998) 93, pp. 165-176). It has been revealed that RANKL is a membraneprotein produced by osteoblasts/stromal cells, its expression beingregulated by a bone resorption factor, RANKL induces differentiation ofosteoclast precursor cells into multinucleated osteoclasts, and the like(Proceedings of the National Academy of Science of the United States ofAmerica, (1998) 95, pp. 3597-3602, Journal of Bone and Mineral Research,(1998) 23, S222). Further, knockout mice devoid of RANKL have been foundto develop an osteopetrosis-like disease, and therefore, RANKL has beenproved to be a physiological osteoclast differentiation-inducing factor(Nature, (1999) 397, pp. 315-323).

As drugs for treating bone metabolism diseases or shortening theduration of treatment, bisphosphonates, active vitamin D₃, calcitoninand derivatives thereof, hormone preparations such as estradiol, SERMs(selective estrogen receptor modulators), ipriflavone, vitamin K₂(menatetrenone), PTH (parathyroid hormone) preparations, calciumpreparations and the like are used. However, these drugs do not alwaysexhibit a satisfactory therapeutic effect and the development of anagent with a more potent therapeutic effect has been demanded.

The cell membranes of immune cells are covered with a dense coating ofvarious glycans, such as sialic acid, which are recognized by variousglycan-binding proteins. Sialic-acid-binding immunoglobulin-like lectins(hereinafter referred to as “siglecs”) are a family of type I membraneproteins which recognize sialylated glycans and bind thereto. Manysiglecs are expressed on the cell membranes of immune cells andrecognize sialic acid similarly present on the cell membranes of immunecells and regulate cell interaction or cell function and are consideredto be involved in the immune response (Nature Reviews Immunology, (2007)7, pp. 255-266). However, there are also a lot of siglec molecules whosephysiological functions have not been elucidated yet. Siglec-15(Sialic-acid binding immunoglobulin-like lectin 15) is a molecule whichhas been newly reported to belong to the Siglecs (Glycobiology, (2007)17, pp. 838-846) and is identical to a molecule called CD33L3 (CD33molecule-like 3). This molecule is highly evolutionarily conserved fromfish to humans and has been found to be strongly expressed in dendriticcells and/or macrophages of human spleen and lymph nodes. Further, as aresult of a binding test using a sialic acid probe, it has also beenfound that human Siglec-15 binds to Neu5Acα2-6GalNAc, and that mouseSiglec-15 binds to Neu5Acα2-3Galβ1-4Glc in addition to Neu5Acα2-6GalNAc(Glycobiology, (2007) 17, pp. 838-846). Until recently, thephysiological role of Siglec-15 was not revealed, however, it has beenreported that the expression of Siglec-15 increases with thedifferentiation and maturation of osteoclasts, and the differentiationof osteoclasts is inhibited by decreasing the expression of Siglec-15 byRNA interference (WO 2007/093042). However, the effect of ananti-Siglec-15 antibody on osteoclast differentiation has not beenelucidated yet.

DISCLOSURE OF THE INVENTION Problems that the Invention is to Solve

An object of the invention is to provide: a gene which is specificallyexpressed in various forms of abnormal bone metabolism such as bonedestruction which are seen in osteoporosis, rheumatoid arthritis, cancermetastasis to bone or the like; a substance which inhibits thedifferentiation and maturation of osteoclasts and the activity thereof;a novel method for screening a therapeutic and/or preventive agent forabnormal bone metabolism; a substance which inhibits the differentiationand maturation of osteoclasts and the activity thereof; and atherapeutic and/or preventive agent for abnormal bone metabolism.

Means for Solving the Problems

The present inventors studied to elucidate the mechanism of osteoclastdifferentiation, maturation and activation in order to find a substancehaving a therapeutic and/or preventive effect on abnormal bonemetabolism. As a result, they found that the expression of the Siglec-15gene increases with the differentiation and maturation of osteoclastsand also found that the differentiation of osteoclasts is inhibited byan antibody which specifically binds to Siglec-15, and thus, theinvention has been completed.

That is, the invention includes the following inventions.

(1) An antibody which specifically recognizes one or more polypeptidescomprising an amino acid sequence described in any one of the following(a) to (i) and inhibits osteoclast formation and/or osteoclastic boneresorption, or a functional fragment of the antibody:

(a) an amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing;

(b) an amino acid sequence comprising amino acid residues 21 to 328 ofthe amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing;

(c) an amino acid sequence comprising amino acid residues 1 to 260 ofthe amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing;

(d) an amino acid sequence comprising amino acid residues 21 to 260 ofthe amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing;

(e) an amino acid sequence represented by SEQ ID NO: 4 in the SequenceListing;

(f) an amino acid sequence comprising amino acid residues 21 to 341 ofthe amino acid sequence represented by SEQ ID NO: 4 in the SequenceListing;

(g) an amino acid sequence comprising amino acid residues 1 to 258 ofthe amino acid sequence represented by SEQ ID NO: 4 in the SequenceListing;

(h) an amino acid sequence comprising amino acid residues 21 to 258 ofthe amino acid sequence represented by SEQ ID NO: 4 in the SequenceListing; and

(i) an amino acid sequence including substitution, deletion or additionof one or several amino acid residues in the amino acid sequencedescribed in (a) to (h).

(2) An antibody which specifically recognizes one or more polypeptidescomprising an amino acid sequence encoded by a nucleotide sequencedescribed in any one of the following (j) to (n) and inhibits osteoclastformation and/or osteoclastic bone resorption, or a functional fragmentof the antibody:

(j) a nucleotide sequence represented by SEQ ID NO: 1;

(k) a nucleotide sequence represented by SEQ ID NO: 3;

(l) a nucleotide sequence represented by SEQ ID NO: 19;

(m) a nucleotide sequence represented by SEQ ID NO: 43; and

(n) a nucleotide sequence of a polynucleotide which hybridizes to apolynucleotide comprising a nucleotide sequence complementary to thenucleotide sequence described in (j) to (m) under stringent conditions.

(3) The antibody or a functional fragment of the antibody according to(1) or (2) which inhibits the process of cell fusion of osteoclasts.

(4) The antibody or a functional fragment of the antibody according toany one of (1) to (3) which inhibits osteoclast formation induced byTNF-α.

(5) The antibody or a functional fragment of the antibody according toany one of (1) to (4) which inhibits in vitro osteoclast formation at aconcentration of 30 μg/ml or less.

(6) The antibody or a functional fragment of the antibody according to(5) which inhibits in vitro osteoclast formation at a concentration of 3μg/ml or less.

(7) The antibody or a functional fragment of the antibody according to(6) which inhibits in vitro osteoclast formation at a concentration of 1μg/ml or less.

(8) The antibody or a functional fragment of the antibody according to(7) which inhibits in vitro osteoclast formation at a concentration offrom 63 ng/ml to 1 μg/ml.

(9) The antibody or a functional fragment of the antibody according toany one of (1) to (4) which inhibits osteoclastic bone resorption.

(10) The antibody or a functional fragment of the antibody according to(9) which inhibits in vitro osteoclastic bone resorption at aconcentration of 3 μg/ml or less.

(11) The antibody or a functional fragment of the antibody according to(10) which inhibits in vitro osteoclastic bone resorption at aconcentration of from 0.3 μg/ml to 3 μg/ml.

(12) The antibody or a functional fragment of the antibody according toany one of (1) to (11) which is obtained by a method comprising thefollowing steps 1) and 2):

1) a step of producing an antibody which specifically recognizes any oneor more sequences of the amino acid sequences described in any one ofthe following (a) to (i):

(a) an amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing;

(b) an amino acid sequence comprising amino acid residues 21 to 328 ofthe amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing;

(c) an amino acid sequence comprising amino acid residues 1 to 260 ofthe amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing;

(d) an amino acid sequence comprising amino acid residues 21 to 260 ofthe amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing;

(e) an amino acid sequence represented by SEQ ID NO: 4 in the SequenceListing;

(f) an amino acid sequence comprising amino acid residues 21 to 341 ofthe amino acid sequence represented by SEQ ID NO: 4 in the SequenceListing;

(g) an amino acid sequence comprising amino acid residues 1 to 258 ofthe amino acid sequence represented by SEQ ID NO: 4 in the SequenceListing;

(h) an amino acid sequence comprising amino acid residues 21 to 258 ofthe amino acid sequence represented by SEQ ID NO: 4 in the SequenceListing; and

(i) an amino acid sequence including substitution, deletion or additionof one or several amino acid residues in the amino acid sequencedescribed in (a) to (h); and

2) a step of screening an antibody which exhibits an inhibitory activityagainst osteoclast formation and/or an inhibitory activity against boneresorption.

(13) The antibody or a functional fragment of the antibody according toany one of (1) to (11) which is obtained by a method comprising thefollowing steps 1) and 2):

1) a step of producing an antibody which specifically recognizes one ormore polypeptides comprising an amino acid sequence encoded by anucleotide sequence described in any one of the following (j) to (n):

(j) a nucleotide sequence represented by SEQ ID NO: 1;

(k) a nucleotide sequence represented by SEQ ID NO: 3;

(l) a nucleotide sequence represented by SEQ ID NO: 19;

(m) a nucleotide sequence represented by SEQ ID NO: 43; and

(n) a nucleotide sequence of a polynucleotide which hybridizes to apolynucleotide comprising a nucleotide sequence complementary to thenucleotide sequence described in (j) to (m) under stringent conditions;and

2) a step of screening an antibody which exhibits an inhibitory activityagainst osteoclast formation and/or an inhibitory activity against boneresorption.

(14) The antibody or a functional fragment of the antibody according toany one of (1) to (13), characterized in that the antibody is amonoclonal antibody.

(15) The antibody or a functional fragment of the antibody according to(14), characterized by having the same epitope specificity as anantibody produced by hybridoma #32A1 (FERM BP-10999).

(16) The antibody or a functional fragment of the antibody according to(14), characterized by competing with an antibody produced by hybridoma#32A1 (FERM BP-10999).

(17) The antibody or a functional fragment of the antibody according to(14), characterized in that the antibody is an antibody produced byhybridoma #32A1 (FERM BP-10999).

(18) The antibody or a functional fragment of the antibody according to(14), characterized by having the same epitope specificity as anantibody produced by hybridoma #41B1 (FERM BP-11000).

(19) The antibody or a functional fragment of the antibody according to(14), characterized by competing with an antibody produced by hybridoma#41B1 (FERM BP-11000).

(20) The antibody or a functional fragment of the antibody according to(14), characterized in that the antibody is an antibody produced byhybridoma #41B1 (FERM BP-11000).

(21) The antibody or a functional fragment of the antibody according toany one of (1) to (20), characterized in that the antibody is a chimericantibody.

(22) The antibody or a functional fragment of the antibody according toany one of (1) to (21), characterized in that the antibody is humanized.

(23) The antibody or a functional fragment of the antibody according toany one of (1) to (16), (18) and (19), characterized in that theantibody is a human antibody.

(24) The antibody or a functional fragment of the antibody according toany one of (1) to (23), characterized in that the antibody is an IgGantibody.

(25) The functional fragment of the antibody according to any one of (1)to (24) which is selected from the group consisting of Fab, F(ab′)2,Fab′ and Fv.

(26) The antibody according to any one of (1) to (16), (18) and (19),characterized by being an scFv.

(27) A pharmaceutical composition characterized by comprising at leastone of the antibodies or functional fragments of the antibodiesaccording to (1) to (26).

(28) The pharmaceutical composition according to (27), characterized bybeing a therapeutic and/or preventive agent for abnormal bonemetabolism.

(29) A pharmaceutical composition for treating and/or preventingabnormal bone metabolism characterized by comprising at least one of theantibodies or functional fragments of the antibodies according to (1) to(26) and at least one member selected from the group consisting ofbisphosphonates, active vitamin D₃, calcitonin and derivatives thereof,hormone preparations such as estradiol, SERMs (selective estrogenreceptor modulators), ipriflavone, vitamin K₂ (menatetrenone), calciumpreparations, PTH (parathyroid hormone) preparations, nonsteroidalanti-inflammatory agents, soluble TNF receptor preparations, anti-TNF-αantibodies or functional fragments of the antibodies, anti-PTHrP(parathyroid hormone-related protein) antibodies or functional fragmentsof the antibodies, IL-1 receptor antagonists, anti-IL-6 receptorantibodies or functional fragments of the antibodies, anti-RANKLantibodies or functional fragments of the antibodies and OCIF(osteoclastogenesis inhibitory factor).

(30) The pharmaceutical composition according to (28) or (29), whereinthe abnormal bone metabolism is selected from the group consisting ofosteoporosis, bone destruction accompanying rheumatoid arthritis,cancerous hypercalcemia, bone destruction accompanying multiple myelomaor cancer metastasis to bone, giant cell tumor, tooth loss due toperiodontitis, osteolysis around a prosthetic joint, bone destruction inchronic osteomyelitis, Paget's disease of bone, renal osteodystrophy andosteogenesis imperfecta.

(31) The pharmaceutical composition according to (28), characterized inthat the abnormal bone metabolism is osteoporosis, bone destructionaccompanying rheumatoid arthritis or bone destruction accompanyingcancer metastasis to bone.

(32) The pharmaceutical composition according to (29), characterized inthat the osteoporosis is postmenopausal osteoporosis, senileosteoporosis, secondary osteoporosis due to the use of a therapeuticagent such as a steroid or an immunosuppressant, or osteoporosisaccompanying rheumatoid arthritis.

(33) A method of treating and/or preventing abnormal bone metabolismcharacterized by administering at least one of the antibodies orfunctional fragments of the antibodies according to (1) to (26).

(34) A method of treating and/or preventing abnormal bone metabolismcharacterized by simultaneously or successively administering at leastone of the antibodies or functional fragments of the antibodiesaccording to (1) to (26) and at least one member selected from the groupconsisting of bisphosphonates, active vitamin D₃, calcitonin andderivatives thereof, hormone preparations such as estradiol, SERMs(selective estrogen receptor modulators), ipriflavone, vitamin K₂(menatetrenone), calcium preparations, PTH (parathyroid hormone)preparations, nonsteroidal anti-inflammatory agents, soluble TNFreceptor preparations, anti-TNF-α antibodies or functional fragments ofthe antibodies, anti-PTHrP (parathyroid hormone-related protein)antibodies or functional fragments of the antibodies, IL-1 receptorantagonists, anti-IL-6 receptor antibodies or functional fragments ofthe antibodies, anti-RANKL antibodies or functional fragments of theantibodies and OCIF (osteoclastogenesis inhibitory factor).

(35) The treatment and/or prevention method according to (33) or (34),characterized in that the abnormal bone metabolism is osteoporosis, bonedestruction accompanying rheumatoid arthritis or bone destructionaccompanying cancer metastasis to bone.

(36) The treatment and/or prevention method according to (35),characterized in that the osteoporosis is postmenopausal osteoporosis,senile osteoporosis, secondary osteoporosis due to the use of atherapeutic agent such as a steroid or an immunosuppressant, orosteoporosis accompanying rheumatoid arthritis.

(37) Hybridoma #32A1 (FERM BP-10999).

(38) Hybridoma #41B1 (FERM BP-11000).

Advantage of the Invention

According to the invention, a therapeutic and/or preventive agent forabnormal bone metabolism whose mechanism of action is to inhibit thedifferentiation and maturation of osteoclasts and the activity thereofcan be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows graphs depicting an expression profile analysis for RANK,RANKL, cathepsin K, and TRAP genes in human giant cell tumor tissues.

FIG. 2 shows a graph depicting an expression profile analysis for theSiglec-15 gene in human giant cell tumor tissues.

FIG. 3 shows a graph depicting a change in the expression level of theSiglec-15 gene when osteoclast differentiation was induced from RAW264.7 or mouse bone marrow cells.

FIG. 4 shows graphs depicting the expression of cathepsin K and TRAPgenes accompanying osteoclast differentiation of RAW 264.7 cells.

FIG. 5 shows a graph depicting the expression of the Siglec-15 geneaccompanying osteoclast differentiation of RAW 264.7 cells.

FIG. 6 shows the results of detecting a change in the expression ofmouse Siglec-15-His in 293F cells with culture time bySDS-polyacrylamide electrophoresis and Western blotting using ananti-6-His-HRP antibody.

FIG. 7 shows the results of detecting a change in the expression ofmouse Siglec-15-Fc in 293F cells with culture time by SDS-polyacrylamideelectrophoresis and Western blotting using an anti-human IgG-Fc-HRPantibody.

FIG. 8 shows the results of evaluating the purity of mouse Siglec-15-Hispurified by HisTrap HP column chromatography and Resource Q columnchromatography through SDS-polyacrylamide electrophoresis and silverstaining.

FIG. 9 shows the results of detecting the behavior of mouseSiglec-15-His purified by HisTrap HP column chromatography and ResourceQ column chromatography through SDS-polyacrylamide electrophoresis andWestern blotting using an anti-V5-HRP antibody.

FIG. 10 shows the results of evaluating the purity of mouse Siglec-15-Fcpurified by HiTrap Protein A column chromatography throughSDS-polyacrylamide electrophoresis and silver staining.

FIG. 11 shows the results of confirming that a purified anti-mouseSiglec-15 polyclonal antibody binds not only to Siglec-15-Fc but also toSiglec-15-His by SDS-polyacrylamide electrophoresis and Western blottingusing an anti-mouse Siglec-15 polyclonal antibody and an anti-rabbitIgG-HRP antibody.

FIG. 12 shows a chromatogram of an anti-mouse Siglec-15 polyclonalantibody purified with an affinity column having mouse Siglec-15-Fcimmobilized thereon.

FIG. 13 shows the results of evaluating the purity of mouse Siglec-15-Fcpurified by chromatography using an affinity column having mouseSiglec-15-Fc immobilized thereon.

FIG. 14 a and FIG. 14 b show chromatograms of anti-mouse Siglec-15polyclonal antibodies purified with a Superose 6 gel filtration column.

FIG. 15 shows the results of testing the effect of the addition of anaffinity-purified anti-mouse Siglec-15 polyclonal antibody on osteoclastdifferentiation (stimulation with RANKL) of mouse bone marrownonadherent cells (N=3).

FIG. 16 shows the results of testing the effect of the addition of a gelfiltration-purified anti-mouse Siglec-15 polyclonal antibody onosteoclast differentiation (stimulation with RANKL) of mouse bone marrownonadherent cells based on the enzymatic activity of TRAP (N=3).

FIG. 17 shows the results of testing neutralization by an antigen ofinhibition of osteoclast differentiation (stimulation with RANKL) ofmouse bone marrow nonadherent cells by the addition of an anti-mouseSiglec-15 polyclonal antibody based on the enzymatic activity of TRAP(N=3).

FIG. 18 shows the results of testing the effect of the addition of ananti-mouse Siglec-15 polyclonal antibody on osteoclast differentiation(stimulation with TNF-α) of mouse bone marrow nonadherent cells based onthe enzymatic activity of TRAP (N=3).

FIG. 19 shows photomicrographs used for evaluating the effect of theaddition of an anti-mouse Siglec-15 polyclonal antibody on osteoclastdifferentiation (stimulation with TNF-α) of mouse bone marrownonadherent cells by TRAP staining.

FIG. 20 shows graphs depicting, by the enzymatic activity of TRAP, theinhibition of osteoclast differentiation (stimulation with activevitamin D₃) from mouse bone marrow cells by the addition of ananti-mouse Siglec-15 polyclonal antibody (N=6).

FIG. 21 shows photomicrographs depicting, by TRAP staining, theinhibition of giant osteoclast formation (stimulation with activevitamin D₃) from mouse bone marrow cells by the addition of ananti-mouse Siglec-15 polyclonal antibody.

FIG. 22 shows photomicrographs depicting, by TRAP staining, theinhibition of giant osteoclast formation (stimulation with human RANKL)from mouse bone marrow cells by the addition of an anti-mouse Siglec-15polyclonal antibody.

FIG. 23 shows photomicrographs depicting, by TRAP staining, theinhibition of giant osteoclast formation (stimulation with human RANKL)from RAW 264.7 cells by the addition of an anti-mouse Siglec-15polyclonal antibody and cancellation of the inhibitory effect by solubleSiglec-15.

FIG. 24 shows the results of testing the binding of a rat anti-mouseSiglec-15 monoclonal antibody to a plate having mouse Siglec-15-Fcimmobilized thereon by an ELISA method. The symbol (♦) denotes #1A1antibody, the symbol (▪) denotes #3A1 antibody, the symbol (▴) denotes#8A1 antibody, the symbol (x) denotes #24A1 antibody, the symbol ()denotes #32A1 antibody, the symbol (◯) denotes #34A1 antibody, thesymbol (+) denotes #39A1 antibody, the symbol (−) denotes #40A1antibody, the symbol (

) denotes #41B1 antibody, the symbol (⋄) denotes #61A1 antibody, and thesymbol (□) denotes control IgG.

FIG. 25 shows the results of testing the effect of the addition of ananti-mouse Siglec-15 monoclonal antibody (#3A1, #8A1, or #32A1) onosteoclast differentiation (stimulation with RANKL) of mouse bone marrownonadherent cells. The rat control IgG in the figure is a negativecontrol common to FIGS. 25 and 26.

FIG. 26 shows the results of testing the effect of the addition of ananti-mouse Siglec-15 monoclonal antibody (#34A1, #39A1, or #40A1) onosteoclast differentiation (stimulation with RANKL) of mouse bone marrownonadherent cells. The rabbit anti-mouse Siglec-15 polyclonal antibodyNo. 3 in the figure is a positive control common to FIGS. 25 and 26.

FIG. 27 shows graphs depicting a change in the expression of cathepsinK, TRAP or the Siglec-15 gene when osteoclast differentiation wasinduced from normal human osteoclast precursor cells.

FIG. 28 shows the results of examining the purity of human Siglec-15-Hispurified by HisTrap HP column chromatography and Resource Q columnchromatography through SDS-polyacrylamide electrophoresis.

FIG. 29 shows the results of examining the purity of human Siglec-15-Fcpurified by Protein A column chromatography through SDS-polyacrylamideelectrophoresis.

FIG. 30 a, FIG. 30 b, and FIG. 30 c show chromatograms of anti-humanSiglec-15 polyclonal antibodies purified using an affinity column havinghuman Siglec-15-Fc immobilized thereon.

FIG. 31 shows photomicrographs depicting, by TRAP staining, theinhibition of giant osteoclast formation from normal human osteoclastprecursor cells by the addition of an anti-human Siglec-15 polyclonalantibody.

FIG. 32 shows the results of evaluating the effect of the addition of ananti-human Siglec-15 polyclonal antibody on multinucleated osteoclastformation from normal human osteoclast precursor cells by counting thenumber of TRAP-positive cells having 5 or more nuclei with an invertedmicroscope.

FIG. 33 shows the results of testing the binding of a rat anti-mouseSiglec-15 monoclonal antibody to a plate having human Siglec-15-Fcimmobilized thereon by an ELISA method. The symbol (♦) denotes #1A1antibody, the symbol (▪) denotes #3A1 antibody, the symbol (▴) denotes#8A1 antibody, the symbol (x) denotes #24A1 antibody, the symbol ()denotes #32A1 antibody, the symbol (◯) denotes #34A1 antibody, thesymbol (+) denotes #39A1 antibody, the symbol (−) denotes #40A1antibody, the symbol (

) denotes #41B1 antibody, the symbol (⋄) denotes #61A1 antibody, and thesymbol (□) denotes control IgG.

FIG. 34 shows photomicrographs depicting, by TRAP staining, theinhibition of giant osteoclast formation from normal human osteoclastprecursor cells by the addition of a rat anti-mouse Siglec-15 monoclonalantibody.

FIG. 35 shows photomicrographs depicting, by TRAP staining, theinhibition of giant osteoclast formation from normal human osteoclastprecursor cells by the addition of a rat anti-mouse Siglec-15 monoclonalantibody (#32A1 antibody).

FIG. 36 shows a graph depicting the inhibition of the bone resorptionactivity of normal human osteoclasts by the addition of a rat anti-mouseSiglec-15 monoclonal antibody (#32A1 antibody) (N=6).

BEST MODE FOR CARRYING OUT THE INVENTION

The term “gene” as used herein includes not only DNA, but also mRNA,cDNA and cRNA.

The term “polynucleotide” as used herein is used in the same meaning asa nucleic acid and also includes DNA, RNA, probes, oligonucleotides andprimers.

The terms “polypeptide” and “protein” as used herein are used withoutdistinction.

The term “RNA fraction” as used herein refers to a fraction containingRNA.

The term “cell” as used herein also includes cells in an animalindividual and cultured cells.

The term “Siglec-15” as used herein is used in the same meaning asSiglec-15 protein.

The term “osteoclast formation” as used herein is used in the samemeaning as “osteoclast differentiation” or “osteoclast maturation”.

The term “functional fragment of an antibody” as used herein refers to apartial fragment of an antibody having an antigen-binding activity andincludes Fab, F(ab′)2, scFv and the like. The term also encompasses Fab′which is a monovalent fragment in a variable region of an antibodyobtained by treating F(ab′)2 under reducing conditions. However, theterm is not limited to these molecules as long as the fragment has abinding affinity for an antigen. Further, these functional fragmentsinclude not only a fragment obtained by treating a full-length moleculeof an antibody protein with an appropriate enzyme, but also a proteinproduced in an appropriate host cell using a genetically modifiedantibody gene.

The term “epitope” as used herein refers to a partial peptide ofSiglec-15 to which a specific anti-Siglec-15 antibody binds. Theabove-mentioned epitope which is a partial peptide of Siglec-15 can bedetermined by methods well known to those skilled in the art such as animmunoassay. Alternatively, for example, the following method can beemployed. Various partial structures of Siglec-15 are produced. In theproduction of the partial structures, a known oligopeptide synthesistechnique can be used. For example, a series of polypeptides havingappropriately reduced lengths obtained by sequentially shorteningSiglec-15 from the C terminus or N terminus are produced using a geneticrecombination technique known to those skilled in the art. Thereafter,the reactivity of an antibody against these polypeptides is examined anda recognition site is roughly determined. Then, peptides having shorterlengths are synthesized and the reactivity with these peptides isexamined, whereby the epitope can be determined. If a secondanti-Siglec-15 antibody binds to a partial peptide to which a firstanti-Siglec-15 antibody binds, it can be determined that the firstantibody and the second antibody share the same epitope. Further, byconfirming that the second anti-Siglec-15 antibody competes with thefirst anti-Siglec-15 antibody for the binding to Siglec-15 (that is, thesecond antibody inhibits the binding between Siglec-15 and the firstantibody), it can be determined that the first antibody and the secondantibody share the same epitope even if the specific epitope sequencehas not been determined. Further, when the first antibody and the secondantibody bind to the same epitope and also the first antibody has aspecial effect such as an antigen-neutralizing activity, the secondantibody can be expected to have the same activity.

The phrase “hybridization is performed under stringent conditions” asused herein refers to hybridization being performed under the conditionsunder which identification can be effected by performing hybridizationat 68° C. in a commercially available hybridization solution, ExpressHybHybridization Solution (manufactured by Clontech, Inc.) or performinghybridization at 68° C. in the presence of 0.7 to 1.0 M NaCl using afilter having DNA immobilized thereon, followed by performing washing at68° C. using 0.1 to 2×SSC solution (1×SSC solution is composed of 150 mMNaCl and 15 mM sodium citrate) or under conditions equivalent thereto.

1. Siglec-15

The present inventors found that the Siglec-15 gene is specificallyexpressed in giant cell tumors and also found that the expression levelof the Siglec-15 gene increases when a monocyte-derived cell linedifferentiates into osteoclasts.

As for Siglec-15 to be used in the invention, Siglec-15 is directlypurified from monocytes or bone marrow cells of human, non-human mammal(such as guinea pig, rat, mouse, rabbit, pig, sheep, cattle or monkey)or chicken and used, or a cell membrane fraction of the above-mentionedcells is prepared and can be used. Further, Siglec-15 can be obtained byin vitro synthesis thereof or production thereof in a host cell throughgenetic engineering. In the genetic engineering, specifically, Siglec-15cDNA is integrated into a vector capable of expressing Siglec-15 cDNA,and Siglec-15 is synthesized in a solution containing enzymes,substrates, and energy substances required for transcription andtranslation, or another prokaryotic or eucaryotic host cell istransformed to express Siglec-15, whereby the protein can be obtained.

The nucleotide sequence of human Siglec-15 cDNA has been registered inGenBank with an accession number of NM_(—)213602 and is represented bySEQ ID NO: 1 in the Sequence Listing, and its amino acid sequence isrepresented by SEQ ID NO: 2 in the Sequence Listing. The nucleotidesequence of mouse Siglec-15 cDNA has been registered in GenBank with anaccession number of XM_(—)884636 and is represented by SEQ ID NO: 3 inthe Sequence Listing, and its amino acid sequence is represented by SEQID NO: 4 in the Sequence Listing. Mature human Siglec-15 from which thesignal sequence has been removed corresponds to an amino acid sequencecomposed of amino acid residues 21 to 328 of the amino acid sequencerepresented by SEQ ID NO: 2. Further, mouse Siglec-15 from which thesignal sequence has been removed corresponds to an amino acid sequencecomposed of amino acid residues 21 to 341 of the amino acid sequencerepresented by SEQ ID NO: 4. Incidentally, Siglec-15 is sometimes calledCD33 antigen-like 3, CD33 molecule-like 3, CD33-like 3 or CD33L3, andall of these represent the same molecule.

Siglec-15 cDNA can be obtained by, for example, a so-called PCR methodin which a polymerase chain reaction (hereinafter referred to as “PCR”)is performed using a cDNA library expressing Siglec-15 cDNA as atemplate and primers which specifically amplify Siglec-15 cDNA (Saiki,R. K., et al., Science, (1988) 239, 487-49).

Further, a polynucleotide which hybridizes to a polynucleotide composedof a nucleotide sequence complementary to at least one nucleotidesequence selected from the nucleotide sequences represented by SEQ IDNOS: 1 and 3 in the Sequence listing under stringent conditions andwhich encodes a protein having a biological activity comparable to thatof Siglec-15 is also included in Siglec-15 cDNA. Further, apolynucleotide which is a splicing variant transcribed from the human ormouse Siglec-15 locus or a polynucleotide which hybridizes thereto understringent conditions and encodes a protein having a biological activitycomparable to that of Siglec-15 is also included in Siglec-15 cDNA.

Further, a protein which is composed of an amino acid sequence includingsubstitution, deletion or addition of one or several amino acids in atleast one amino acid sequence selected from the amino acid sequencesrepresented by SEQ ID NOS: 2 and 4 in the Sequence Listing or the aminoacid sequence from which the signal sequence has been removed and has abiological activity comparable to that of Siglec-15 is also included inSiglec-15. Further, a protein which is composed of an amino acidsequence encoded by a splicing variant transcribed from the human ormouse Siglec-15 locus or an amino acid sequence including substitution,deletion or addition of one or several amino acids therein and has abiological activity comparable to that of Siglec-15 is also included inSiglec-15.

2. Detection of Abnormal Bone Metabolism

An analysis of the expression level of the Siglec-15 gene in a group oftest samples from various human bone tissues showed that the expressionlevel of the gene significantly increases in giant cell tumor (GCT)which is a bone tumor with a large number of osteoclast-likemultinucleated giant cells arising and is characterized by clinicalfindings of osteolytic bone destruction (Bullough et al., Atlas ofOrthopedic Pathology 2nd edition, pp. 17.6-17.8, Lippincott Williams &Wilkins Publishers (1992)).

It was also found that the expression level of the Siglec-15 geneincreases when a monocyte-derived cell line is differentiated intoosteoclasts.

Accordingly, Siglec-15 is considered to be associated with humanpathology such as GCT in which bone resorption is increased. In otherwords, measurement of the expression level of the Siglec-15 gene and/orSiglec-15 in each cell and/or each tissue enables determination of thestate of abnormal bone metabolism accompanied by overexpression ofSiglec-15. Examples of the abnormal bone metabolism as used hereininclude, but are not limited to, osteoporosis (postmenopausalosteoporosis, senile osteoporosis, secondary osteoporosis due to the useof a therapeutic agent such as a steroid or an immunosuppressant, orosteoporosis accompanying rheumatoid arthritis), bone destructionaccompanying rheumatoid arthritis, cancerous hypercalcemia, bonedestruction accompanying multiple myeloma or cancer metastasis to bone,giant cell tumor, tooth loss due to periodontitis, osteolysis around aprosthetic joint, bone destruction in chronic osteomyelitis, Paget'sdisease of bone, renal osteodystrophy and osteogenesis imperfecta.

In the invention, the “test sample” to be used for examining theexpression level of the Siglec-15 gene and/or Siglec-15 refers to asample from a tissue of bone marrow, bone, prostate, testis, penis,bladder, kidney, oral cavity, pharynx, lip, tongue, gingiva,nasopharynx, esophagus, stomach, small intestine, large intestine,colon, liver, gallbladder, pancreas, nose, lung, soft tissue, skin,breast, uterus, ovary, brain, thyroid, lymph node, muscle, fat tissue orthe like, or blood, a body fluid or an excretion or the like, however,in the invention, blood or bone marrow is more preferred.

3. Method of Screening Substance which Inhibits Differentiation intoOsteoclasts

As one embodiment of the invention, a method of screening a substancewhich inhibits differentiation into osteoclasts by measuring theexpression level of the Siglec-15 gene and/or Siglec-15 can beexemplified.

As another embodiment of the invention, a method of screening asubstance which has a therapeutic effect and/or preventive effect onabnormal bone metabolism by identifying a substance which inhibits theactivity of Siglec-15 of inducing differentiation into matureosteoclasts, can be exemplified.

The “test substance” refers to a substance to be used for examining theactivity of inhibiting differentiation into osteoclasts by the screeningmethod of the invention. Examples of the test substance include acompound, a microbial metabolite, an extract from a plant or animaltissue, a derivative thereof, and a mixture thereof. Further, a nucleicacid designed to decrease the expression level of Siglec-15 or aderivative thereof (such as an antisense oligonucleotide, a ribozyme,dsRNA or siRNA) can also be used as the test substance. The dose orconcentration of the test substance may be appropriately set or aplurality of doses may be set by, for example, preparing dilution seriesthereof. The test substance can be administered in an appropriate statesuch as a solid or a liquid, and may be dissolved in an appropriatebuffer or a stabilizer or the like may be added thereto. In the case ofa screening method using cultured cells, the test substance is added toa medium and cells can be cultured therein. In the case where the testsubstance is added to a medium, the test substance may be added at theinitiation of the culturing or in the middle of the culturing, and thenumber of addition operations is not limited to one. The period ofculturing in the presence of a test substance may be appropriately set,however, it is preferably from 30 minutes to 2 weeks, more preferablyfrom 30 minutes to 72 hours. In the case where the test substance isadministered to a mammalian individual, the route of administration,including oral administration, intravenous injection, intraperitonealinjection, transdermal administration, subcutaneous injection and thelike, is suitably determined depending on the physical properties andthe like of the test substance. Incidentally, a suitable time until thetest sample is obtained, after the administration of the test substance,can be selected.

The cultured cells to be used in the screening method of the inventionmay be normal cells, an established cell line, or cells showing abnormalgrowth such as cancer cells as long as they are mammalian cellsexpressing Siglec-15. Examples thereof include, but are not limited to,normal human osteoclast precursor cells (Normal Human Natural OsteoclastPrecursor Cells, available from Sanko Junyaku Co., Ltd., Cat. No.2T-110), mouse monocyte-derived cells RAW 264.7 (ATCC Cat. No. TIB-71),RAW 264 cells (ECACC Cat. No. 85062803), and mouse bone marrow-derivedprimary cultured cells. As an animal species of the cultured cells,human, mouse, or other mammals (guinea pig, rat, rabbit, pig, sheep,cattle, monkey, etc.), chicken and the like are preferred, but thespecies is not limited thereto. Incidentally, as the cultured cells, itis more preferred to use mammalian cells overexpressing Siglec-15, andfor example, RAW 264.7 cells, RAW 264 cells, 293 cells, CHO cells andCOST cells modified by introducing the Siglec-15 gene along with thepromoter region thereof to overexpress Siglec-15, and the like can beexemplified.

The screening method of the invention also includes a method ofdetecting the expression of the Siglec-15 gene in cells of an organ or atissue resected from a mammalian individual after administering a testsubstance to the mammalian individual without using cultured cells. Theorgan or tissue to be used for detecting the expression of the gene maybe any as long as it expresses Siglec-15, however, a tissue associatedwith abnormal bone metabolism is preferred, and a bone tissue and bonemarrow are more preferred. As a mammalian species, a non-human mammalcan be used, and mouse, rat or hamster is preferred, and mouse or rat ismore preferred. As an animal model having abnormal bone metabolism, ananimal having the ovary removed, an animal having the testicle removed,a cancer-bearing animal having tumor cells implanted under the skin,into the skin, left ventricle, bone marrow, vein, abdominal cavity orthe like, an animal having a sciatic nerve removed, an animal model ofadjuvant arthritis, an animal model of collagen-induced arthritis, ananimal model of glucocorticoid-induced osteoporosis, asenescence-accelerated mouse (SAM P6 mouse, Matsushita et al., Am. J.Pathol. 125, 276-283 (1986)), an animal having the thyroid/parathyroidremoved, an animal receiving a continuous infusion of a parathyroidhormone-related peptide (PTHrP), an osteoclastogenesis inhibitory factor(OCIF) knockout mouse (Mizuno et al., Biochem. Biophys. Res. Commun.,(1998) 247, 610-615), an animal with the administration of soluble RANKLor the like can be used. Further, an animal model with tooth loss due toperiodontal disease or an animal modified to overexpress Siglec-15 canalso be used. Further, a test substance selected by screening isadministered to any of the above-mentioned animal models, and eachparameter which can be obtained by the measurement of the number ofmature osteoclasts in a bone tissue, a bone density, a bone strength ora bone morphology, bone metabolism parameters (CTx, NTx, etc.) in bloodand urine or parameters that vary due to abnormal bone metabolism suchas blood calcium levels are measured, whereby the therapeutic effectand/or preventive effect of the test substance on abnormal bonemetabolism can be evaluated.

The cultured cells to be used in the method of the invention may becultured under any conditions as long as the conditions enable thecultured cells to express Siglec-15 without the addition of a testsubstance. For example, there are known culture conditions for thecultured cells, and when the cells express Siglec-15 under theconditions, the cells may be cultured under the conditions. Further, inthe case where the expression of Siglec-15 in an organ or a tissueresected from a mammalian individual is detected, rearing conditions forthe animal may be any as long as the conditions enable the animal toexpress Siglec-15 without the addition of a test substance.

In order to examine the effect of a test substance on the expression ofSiglec-15, there are a method of measuring the expression level of theSiglec-15 gene and a method of measuring the expression level ofSiglec-15 which is a translation product of the Siglec-15 gene. A testsubstance which inhibits the expression of the Siglec-15 gene and/orSiglec-15 is considered to be a substance having a therapeutic effectand/or preventive effect on abnormal bone metabolism, preferablyosteoporosis, or bone destruction accompanying rheumatoid arthritisand/or cancer metastasis to bone.

The measurement of the expression level of the Siglec-15 gene orSiglec-15 in cultured cells can be performed by a Northern analysis, aquantitative PCR method, an ELISA method or the like. In the case wheremammalian cultured cells are used, an appropriate amount of RANKL,TNF-α, M-CSF, active vitamin D₃, or the like is added, as needed, to amedium along with a test substance, and also in a control without theaddition of the test substance, an appropriate amount of RANKL, TNF-α,M-CSF, active vitamin D₃, or the like is added.

Further, an experimental system for measuring the binding amount of anendogenous ligand to Siglec-15 is constructed, and whether or not thebinding of the endogenous ligand to Siglec-15 is inhibited by theaddition of a test substance is evaluated, whereby screening of asubstance which inhibits differentiation into osteoclasts can beperformed.

The respective screening methods will be described in the following (1)to (3).

(1) Method Using Siglec-15 Gene

As the screening method of the invention, for example, there are amethod using mammalian cultured cells and a method using mammalianindividuals, which will be described below, respectively.

(a) Method using mammalian cultured cells

(i) A method including the following steps a) to c):

a) a step of extracting total RNA from mammalian cultured cells culturedin a medium with the addition of a test substance;

b) a step of detecting a difference in the expression level of theSiglec-15 gene between the total RNA obtained in a) and total RNAobtained from mammalian cultured cells cultured without the addition ofthe test substance; and

c) a step of determining the therapeutic and/or preventive effect of thetest substance on abnormal bone metabolism by analyzing the differencein the expression level of the gene described in b).

(ii) A method including the following steps a) to d):

a) a step of extracting total RNA from mammalian cultured cells culturedin a medium with the addition of a test substance;

b) a step of extracting total RNA from mammalian cultured cells culturedin a medium without the addition of the test substance;

c) a step of measuring the expression level of the Siglec-15 gene in thetotal RNA obtained in a) and in the total RNA obtained in b),respectively; and

d) a step of determining the therapeutic and/or preventive effect of thetest substance on abnormal bone metabolism by analyzing a difference inthe expression level of the gene measured in c) between the total RNAobtained in a) and the total RNA obtained in b).

(b) Method using mammalian individuals

(i) A method including the following steps (a) to (c):

a) a step of extracting total RNA from a test sample collected from amammalian individual with the administration of a test substance;

b) a step of detecting a difference in the expression level of theSiglec-15 gene between the total RNA obtained in a) and total RNAobtained from a test sample collected from a mammalian individualwithout the administration of the test substance; and

c) a step of determining the therapeutic and/or preventive effect of thetest substance on abnormal bone metabolism by analyzing the differencein the expression level of the gene described in b).

(ii) A method including the following steps a) to d):

a) a step of extracting total RNA from a test sample collected from amammalian individual with the administration of a test substance;

b) a step of extracting total RNA from a test sample collected from amammalian individual without the administration of the test substance;

c) a step of measuring the expression level of the Siglec-15 gene in thetotal RNA obtained in the step a) and in the total RNA obtained in thestep b), respectively; and

d) a step of determining the therapeutic and/or preventive effect of thetest substance on abnormal bone metabolism by analyzing a difference inthe expression level of the gene described in c).

(2) Method Using Siglec-15

As the screening method utilizing the measurement of the expressionlevel of Siglec-15, there are a method using mammalian cultured cellsand a method using animal individuals each of which includes thefollowing steps.

(a) Method using mammalian cultured cells

(i) A method including the following steps a) and b):

a) a step of measuring the expression level of Siglec-15 in mammaliancultured cells cultured in a medium with the addition of a testsubstance; and

b) a step of determining the therapeutic effect and/or preventive effectof the test substance on abnormal bone metabolism by analyzing adifference between the expression level of the protein measured in a)and the expression level of the protein in mammalian cultured cellscultured in a medium without the addition of the test substance.

(ii) A method including the following steps a) to c):

a) a step of measuring the expression level of Siglec-15 in mammaliancultured cells cultured in a medium with the addition of a testsubstance;

b) a step of measuring the expression level of the protein described ina) in mammalian cultured cells cultured in a medium without the additionof the test substance; and

c) a step of determining the therapeutic effect and/or preventive effectof the test substance on abnormal bone metabolism by detecting adifference between the expression level of the protein measured in a)and the expression level of the protein measured in b).

(iii) A method including the following steps a) to c):

a) a step of immobilizing total protein obtained from mammalian culturedcells cultured in a medium with the addition of a test substance;

b) a step of measuring the expression level of Siglec-15 in theimmobilized protein; and

c) a step of determining the therapeutic effect and/or preventive effectof the test substance on abnormal bone metabolism by analyzing adifference between the expression level of Siglec-15 detected in b) andthe expression level of the protein in total protein obtained frommammalian cultured cells cultured in a medium without the addition ofthe test substance.

(iv) A method including the following steps a) to e):

a) a step of immobilizing total protein obtained from mammalian culturedcells cultured in a medium with the addition of a test substance;

b) a step of immobilizing total protein obtained from mammalian culturedcells cultured in a medium without the addition of the test substance;

c) a step of measuring the expression level of Siglec-15 in theimmobilized protein described in the step a) using an antibody or aligand specifically binding to the protein;

d) a step of measuring the expression level of Siglec-15 in theimmobilized protein described in the step b) using an antibody or aligand specifically binding to the protein; and

e) a step of determining the therapeutic effect and/or preventive effectof the test substance on abnormal bone metabolism by analyzing adifference between the expression level of the protein measured in thestep c) and the expression level of the protein measured in the step d).

(b) Method using mammalian individuals

(i) A method including the following steps a) and b):

a) a step of measuring the expression level of Siglec-15 in a testsample collected from a mammalian individual to whom a test substancehas been administered; and

b) a step of determining the therapeutic effect and/or preventive effectof the test substance on abnormal bone metabolism by analyzing adifference between the expression level of Siglec-15 measured in thestep a) and the expression level of the protein in a test samplecollected from a mammalian individual without the administration of thetest substance.

(ii) A method including the following steps a) to c):

a) a step of measuring the expression level of Siglec-15 in a testsample collected from a mammalian individual, to whom a test substancehas been administered, using an antibody or a ligand specificallybinding to the protein;

b) a step of measuring the expression level of the protein in a testsample collected from a mammalian individual without the administrationof the test substance; and

c) a step of determining the therapeutic effect and/or preventive effectof the test substance on abnormal bone metabolism by analyzing adifference between the expression level of Siglec-15 measured in a) andthe expression level of the protein measured in b).

(iii) A method including the following steps a) to c):

a) a step of immobilizing total protein in a test sample collected froma mammalian individual to whom a test substance has been administered;

b) a step of measuring the expression level of Siglec-15 in theimmobilized protein; and

c) a step of determining the therapeutic and/or preventive effect of thetest substance on abnormal bone metabolism by analyzing a differencebetween the expression level of Siglec-15 detected in b) and theexpression level of the protein in a test sample collected from amammalian individual without the administration of the test substance.

(iv) A method including the following steps a) to e):

a) a step of immobilizing total protein in a test sample collected froma mammalian individual to whom a test substance has been administered;

b) a step of immobilizing total protein in a test sample collected froma mammalian individual without the administration of the test substance;

c) a step of detecting the expression level of Siglec-15 in theimmobilized protein described in the step a) using an antibody or aligand specifically binding to the protein;

d) a step of detecting the expression level of Siglec-15 in theimmobilized protein described in b) using an antibody or a ligandspecifically binding to the protein; and

e) a step of determining the therapeutic effect and/or preventive effectof the test substance on abnormal bone metabolism by analyzing adifference between the expression level of the protein detected in c)and the expression level of the protein detected in d).

(3) Screening Method Using Endogenous Ligand

Screening of a substance which inhibits differentiation into osteoclastscan also be performed by observing whether or not the binding of anendogenous ligand to Siglec-15 is inhibited by the addition of a testsubstance. Examples of a sialylated glycan serving as an endogenousligand for Siglec-15 include Neu5Acα2-6GalNAc binding to human and mouseSiglec-15 and Neu5Acα2-3Galβ1-4Glc binding to mouse Siglec-15. However,the endogenous ligand for Siglec-15 is not limited to these glycans aslong as it has a binding affinity for Siglec-15. These endogenousligands can be labeled with an appropriate tag, radioisotope orfluorescent substance for the purpose of examining the binding thereofto Siglec-15. For example, biotinylated polyacrylamide to which asialylated oligosaccharide such as Neu5Acα2-6GalNAc has been bound canbe used in screening as a probe binding to Siglec-15. As Siglec-15 towhich an endogenous ligand is bound, Siglec-15-expressing cells or amembrane fraction prepared from the cells can be used. Further,Siglec-15 can be subjected to screening after Siglec-15 is isolated fromSiglec-15-expressing cells, followed by purification. As theSiglec-15-expressing cells, any of a cultured cell line expressingSiglec-15, cells caused to transiently or constantly express theSiglec-15 gene by subjecting appropriate cultured cells to geneticengineering, and cells expressing Siglec-15 obtained in vivo can beused. The screening method using such an endogenous ligand can beperformed according to the steps as described below.

(i) A method including the following steps a) and b):

a) a step of adding an endogenous ligand for Siglec-15 and a testsubstance to Siglec-15-expressing cells; and

b) a step of determining the therapeutic and/or preventive effect of thetest substance on abnormal bone metabolism by comparing the bindingamount of the endogenous ligand to the Siglec-15-expressing cellsbetween the case where the test substance was added and the case wherethe test substance was not added.

(ii) A method including the following steps a) to c):

a) a step of preparing a cell membrane fraction of Siglec-15-expressingcells;

b) a step of adding an endogenous ligand for Siglec-15 and a testsubstance to the cell membrane fraction; and

c) a step of determining the therapeutic and/or preventive effect of thetest substance on abnormal bone metabolism by comparing the bindingamount of the endogenous ligand to the cell membrane fraction betweenthe case where the test substance was added and the case where the testsubstance was not added.

(iii) A method including the following steps a) to c):

a) a step of preparing Siglec-15;

b) a step of adding an endogenous ligand for Siglec-15 and a testsubstance to Siglec-15 in a); and

c) a step of determining the therapeutic and/or preventive effect of thetest substance on abnormal bone metabolism by comparing the bindingamount of the endogenous ligand to Siglec-15 between the case where thetest substance was added and the case where the test substance was notadded.

In the case where appropriate cells are caused to express Siglec-15 bygenetic engineering and the resulting Siglec-15 is purified andsubjected to screening, Siglec-15 to be screened can be selected frompolypeptides composed of the amino acid sequences shown in the following(a) to (i):

(a) an amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing;

(b) an amino acid sequence composed of amino acid residues 21 to 328 ofthe amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing;

(c) an amino acid sequence composed of amino acid residues 1 to 260 ofthe amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing;

(d) an amino acid sequence composed of amino acid residues 21 to 260 ofthe amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing;

(e) an amino acid sequence represented by SEQ ID NO: 4 in the SequenceListing;

(f) an amino acid sequence composed of amino acid residues 21 to 341 ofthe amino acid sequence represented by SEQ ID NO: 4 in the SequenceListing;

(g) an amino acid sequence composed of amino acid residues 1 to 258 ofthe amino acid sequence represented by SEQ ID NO: 4 in the SequenceListing;

(h) an amino acid sequence composed of amino acid residues 21 to 258 ofthe amino acid sequence represented by SEQ ID NO: 4 in the SequenceListing; and

(i) an amino acid sequence including substitution, deletion or additionof one or several amino acid residues in the amino acid sequencedescribed in (a) to (h).

Siglec-15 to be screened can also be selected from polypeptides composedof amino acid sequences encoded by the nucleotide sequences shown in thefollowing (j) to (n):

(j) a nucleotide sequence represented by SEQ ID NO: 1;

(k) a nucleotide sequence represented by SEQ ID NO: 3;

(l) a nucleotide sequence represented by SEQ ID NO: 19;

(m) a nucleotide sequence represented by SEQ ID NO: 43; and

(n) a nucleotide sequence of a polynucleotide which hybridizes to apolynucleotide composed of a nucleotide sequence complementary to thenucleotide sequence described in (j) to (m) under stringent conditions.

Further, it is also possible to use a polypeptide obtained by attachingan appropriate tag to any of these polypeptides, or a polypeptide fusedwith a different soluble protein, as a target for screening.Incidentally, the polypeptide composed of the amino acid residues 1 to20 of the amino acid sequence represented by SEQ ID NO: 2 in theSequence Listing corresponds to the signal peptide of human Siglec-15,and the polypeptide composed of the amino acid residues 21 to 260thereof corresponds to the extracellular domain of the mature protein ofhuman Siglec-15. Further, the polypeptide composed of the amino acidresidues 1 to 20 of the amino acid sequence represented by SEQ ID NO: 4in the Sequence Listing corresponds to the signal peptide of mouseSiglec-15, and the polypeptide composed of the amino acid residues 21 to258 thereof corresponds to the extracellular domain of the matureprotein of mouse Siglec-15. Further, the nucleotide sequence representedby SEQ ID NO: 43 encodes the extracellular domain of human Siglec-15encoded by the nucleotide sequence represented by SEQ ID NO: 1, and thenucleotide sequence represented by SEQ ID NO: 19 encodes theextracellular domain of mouse Siglec-15 encoded by the nucleotidesequence represented by SEQ ID NO: 3.

Candidate substances for a substance which inhibits differentiation intoosteoclasts selected by any of the screening methods (1) to (3) can besecondarily evaluated by using the inhibition of tartrate-resistant acidphosphatase (TRAP) activity of osteoclasts as an index as shown inExamples 17, 19 and 20. Further, the secondary evaluation thereof canalso be performed by using the inhibition of formation of TRAP-positivemultinucleated osteoclasts, i.e., the inhibition of cell fusion ofosteoclasts as an index as shown in Examples 19, 21, 22 and 35.

(4) Other Methods

The incidence rate of abnormal bone metabolism over time, the degree ofabnormal bone metabolism, and/or the survival rate, etc. are determinedfor the case where a test substance was administered to a mammalianindividual caused to overexpress Siglec-15 and the case where the testsubstance was not administered thereto. In the case where, in the mammalwith the administration of the test substance, the incidence rate ofabnormal bone metabolism is significantly decreased, the degree ofabnormal bone metabolism is significantly lower, and/or the survivalrate is increased by about 10% or more, preferably about 30% or more,more preferably about 50% or more, the test substance can be selected asa compound having a therapeutic and/or preventive effect on abnormalbone metabolism.

4. Production of Anti-Siglec-15 Antibody

The antibody against Siglec-15 of the invention can be obtained byimmunizing an animal with Siglec-15 or an arbitrary polypeptide selectedfrom the amino acid sequence of Siglec-15, and collecting and purifyingthe antibody produced in vivo according to a common procedure. Thebiological species of Siglec-15 to be used as an antigen is not limitedto human, and an animal can be immunized with Siglec-15 derived from ananimal other than human such as mouse or rat. In this case, by examiningthe cross-reactivity between an antibody binding to the obtainedheterologous Siglec-15 and human Siglec-15, an antibody applicable to ahuman disease can be selected.

Further, a monoclonal antibody can be obtained by fusingantibody-producing cells which produce an antibody against Siglec-15with myeloma cells to establish a hybridoma according to a known method(for example, Kohler and Milstein, Nature, (1975) 256, pp. 495-497;Kennet, R. ed., Monoclonal Antibody, pp. 365-367, Prenum Press, N.Y.(1980)). Siglec-15 to be used as an antigen can be obtained by geneticengineering to cause a host cell to express the Siglec-15 gene.

Specifically, the genetic engineering can be performed as follows. Avector capable of expressing the Siglec-15 gene is produced, and theresulting vector is transfected into a host cell to express the gene,and then, the expressed Siglec-15 is purified. Hereinafter, a method ofobtaining an antibody against Siglec-15 will be specifically described.

(1) Preparation of Antigen

Examples of the antigen to be used for producing the anti-Siglec-15antibody include Siglec-15, a polypeptide composed of a partial aminoacid sequence consisting of at least 6 consecutive amino acid residuesof Siglec-15, and a derivative obtained by adding a given amino acidsequence or carrier thereto. Further, in the section “3. Method ofscreening substance which inhibits differentiation into osteoclasts”,Siglec-15 exemplified as a target for screening can also be exemplifiedas an antigen to be used for producing the anti-Siglec-15 antibody.

Siglec-15 can be used after purifying directly from human tumor tissuesor tumor cells. Further, Siglec-15 can be obtained by synthesizing it invitro or by causing a host cell to produce it by genetic engineering.

In the genetic engineering, specifically, Siglec-15 cDNA is integratedinto a vector capable of expressing Siglec-15 cDNA and Siglec-15 issynthesized in a solution containing enzymes, substrates, and energysubstances required for transcription and translation, or anotherprokaryotic or eucaryotic host cell is transformed to express Siglec-15,whereby the antigen can be obtained.

Further, the antigen can also be obtained as a secretory protein byexpressing a fusion protein obtained by ligating the extracellulardomain of Siglec-15 which is a membrane protein to the constant regionof the antibody in an appropriate host-vector system.

Siglec-15 cDNA can be obtained by, for example, a so-called PCR methodin which a polymerase chain reaction (hereinafter referred to as “PCR”)is performed using a cDNA library expressing Siglec-15 cDNA as atemplate and primers which specifically amplify Siglec-15 cDNA (seeSaiki, R. K., et al., Science, (1988) 239, pp. 487-489).

As the in vitro synthesis system of the polypeptide, for example, theRapid Translation System (RTS) manufactured by Roche Diagnostics, Inc.can be exemplified, but it is not limited thereto.

Examples of the prokaryotic host include Escherichia coli and Bacillussubtilis. In order to transform the host cell with a target gene, thehost cell is transformed using a plasmid vector containing a replicon,i.e., a replication origin derived from a species compatible with thehost, and a regulator sequence. Further, the vector preferably has asequence capable of imposing phenotypic selectivity on the transformedcell.

Examples of the eucaryotic host cell include vertebrate cells, insectcells, and yeast cells. As the vertebrate cells, for example,dihydrofolate reductase-deficient strains (Urlaub, G. and Chasin, L. A.,Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220) of simian COS cells(Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCC CRL-1650), murinefibroblasts NIH3T3 (ATCC No. CRL-1658), and Chinese hamster ovariancells (CHO cells; ATCC: CCL-61); and the like are often used, however,it is not limited thereto.

The thus obtained transformant can be cultured according to a commonprocedure, and by the culturing of the transformant, a targetpolypeptide is produced intracellularly or extracellularly.

A suitable medium to be used for the culturing can be selected fromvarious commonly used culture media depending on the employed host cell.If Escherichia coli is employed, an LB medium supplemented with anantibiotic such as ampicillin or IPMG, if necessary, can be used.

A recombinant protein produced intracellularly or extracellularly by thetransformant through the culturing can be separated and purified by anyof various known separation methods utilizing a physical or chemicalproperty of the protein.

Specific examples of the methods include treatment with a common proteinprecipitant, ultrafiltration, various types of liquid chromatographysuch as molecular sieve chromatography (gel filtration), adsorptionchromatography, ion exchange chromatography and affinity chromatography,dialysis, and a combination thereof.

Further, by attaching six histidine residues to a recombinant protein tobe expressed, the protein can be efficiently purified with a nickelaffinity column. Alternatively, by attaching the IgG Fc region to arecombinant protein to be expressed, the protein can be efficientlypurified with a protein A column.

By combining the above-mentioned methods, a large amount of a targetpolypeptide can be easily produced in high yield and high purity.

(2) Production of Anti-Siglec-15 Monoclonal Antibody

Examples of the antibody specifically binding to Siglec-15 include amonoclonal antibody specifically binding to Siglec-15, and a method ofobtaining the antibody is as described below.

Production of a monoclonal antibody generally requires the followingoperational steps of:

(a) purifying a biopolymer to be used as an antigen;

(b) preparing antibody-producing cells by immunizing an animal byinjection of the antigen, collecting the blood, assaying its antibodytiter and determining when the spleen is to be resected;

(c) preparing myeloma cells (hereinafter referred to as “myeloma”);

(d) fusing the antibody-producing cells with the myeloma;

(e) screening a group of hybridomas producing a target antibody;

(f) dividing the hybridomas into single cell clones (cloning);

(g) optionally, culturing the hybridoma or rearing an animal implantedwith the hybridoma for producing a large amount of a monoclonalantibody;

(h) examining the thus produced monoclonal antibody for biologicalactivity and binding specificity, or assaying the same for propertieswith a labeled reagent; and the like.

Hereinafter, the method of producing a monoclonal antibody will bedescribed in detail following the above steps, however, the method isnot limited thereto, and, for example, antibody-producing cells andmyeloma other than spleen cells may be used.

(a) Purification of Antigen

As the antigen, Siglec-15 prepared by the method as described above or apartial peptide thereof can be used.

Further, a membrane fraction prepared from recombinant cells expressingSiglec-15 or the recombinant cells expressing Siglec-15 themselves, andalso a partial peptide of the protein of the invention chemicallysynthesized by a method known to those skilled in the art can also beused as the antigen.

(b) Preparation of Antibody-Producing Cells

The antigen obtained in step (a) is mixed with an adjuvant such asFreund's complete or incomplete adjuvant, or aluminum potassium sulfateand the resulting mixture is used as an immunogen to immunize anexperimental animal. As the experimental animal, any animal used in aknown hybridoma production method can be used without any trouble.Specifically, for example, mouse, rat, goat, sheep, cattle, horse or thelike can be used. However, from the viewpoint of ease of availability ofmyeloma cells to be fused with the extracted antibody-producing cells,mouse or rat is preferably used as the animal to be immunized.

Further, the strain of mouse or rat to be actually used is notparticularly limited, and in the case of mouse, for example, variouslines such as A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA,FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, and 129 can beused, and in the case of rat, for example, Wistar, Low, Lewis, SpragueDawley, ACI, BN, Fischer and the like can be used.

These mice and rats are commercially available frombreeders/distributors of experimental animals, for example, CLEA Japan,Inc. and Charles River Laboratories Japan, Inc.

Among these, in consideration of fusing compatibility with myeloma cellsdescribed below, in the case of mice, BALB/c strain, and in the case ofrats, Wistar and Low lines are particularly preferred.

Further, in consideration of antigenic homology between humans and mice,it is also preferred to use a mouse having decreased biological functionto remove autoantibodies, that is, a mouse with an autoimmune disease.

The age of the mouse or rat at the time of immunization is preferably 5to 12 weeks of age, more preferably 6 to 8 weeks of age.

In order to immunize an animal with Siglec-15 or a recombinant thereof,for example, a known method is described in detail in, for example,Weir, D. M., Handbook of Experimental Immunology Vol. I. II. III.,Blackwell Scientific Publications, Oxford (1987), Kabat, E. A. andMayer, M. M., Experimental Immunochemistry, Charles C Thomas PublisherSpigfield, Ill. (1964), or the like.

Among these immunization methods, a preferred specific method in theinvention is, for example, as follows.

That is, first, a membrane protein fraction serving as the antigen orcells caused to express the antigen is/are intradermally orintraperitoneally administered to an animal.

However, the combination of both routes of administration is preferredfor increasing the immunization efficiency, and when intradermaladministration is performed in the first half and intraperitonealadministration is performed in the latter half or only at the lastdosing, the immunization efficiency can be particularly increased.

The administration schedule of the antigen varies depending on the typeof animal to be immunized, individual differences or the like. However,in general, the frequency of administration of the antigen is preferably3 to 6 times, more preferably 3 to 4 times, and the dosing interval ispreferably 2 to 6 weeks, more preferably 2 to 4 weeks.

Further, the dose of the antigen varies depending on the type of animalto be immunized, individual differences or the like. However, the doseis generally set to 0.05 to 5 mg, preferably about 0.1 to 0.5 mg.

A booster immunization is performed 1 to 6 weeks, preferably 2 to 4weeks, more preferably 2 to 3 weeks after the administration of theantigen as described above.

The dose of the antigen at the time of performing the boosterimmunization varies depending on the type or size of animal or the like,however, in the case of a mouse, the dose is generally set to 0.05 to 5mg, preferably 0.1 to 0.5 mg, more preferably about 0.1 to 0.2 mg.

Spleen cells or lymphocytes including antibody-producing cells areaseptically removed from the immunized animal 1 to 10 days, preferably 2to 5 days, more preferably 2 to 3 days after the booster immunization.

At this time, the antibody titer is measured, and if an animal having asufficiently increased antibody titer is used as a supply source of theantibody-producing cells, the subsequent procedure can be carried outmore efficiently.

The method of measuring the antibody titer to be used here includes, forexample, an RIA method and an ELISA method, but is not limited thereto.

For example, if an ELISA method is employed, the measurement of theantibody titer in the invention can be carried out according to theprocedures as described below.

First, a purified or partially purified antigen is adsorbed on thesurface of a solid phase such as a 96-well plate for ELISA, and thesurface of the solid phase having no antigen adsorbed thereon is coveredwith a protein unrelated to the antigen such as bovine serum albumin(hereinafter referred to as “BSA”). After washing the surface, thesurface is brought into contact with a serially-diluted sample (forexample, mouse serum) as a primary antibody to allow the antibodycontained in the sample to bind to the antigen.

Further, as a secondary antibody, an antibody labeled with an enzymeagainst a mouse antibody is added and is allowed to bind to the mouseantibody. After washing, a substrate for the enzyme is added and thechange in absorbance which occurs due to color development induced bydegradation of the substrate or the like is measured to calculate theantibody titer.

The separation of the antibody-producing cells from the spleen cells orlymphocytes can be carried out according to a known method (for example,Kohler et al., Nature (1975), 256, p. 495; Kohler et al., Eur. J.Immunol. (1977), 6, p. 511; Milstein et al., Nature (1977), 266, p. 550;Walsh, Nature (1977), 266, p. 495).

For example, in the case of spleen cells, a general method in which theantibody-producing cells are separated by homogenizing the spleen toobtain cells through filtration with a stainless steel mesh andsuspending the cells in Eagle's Minimum Essential Medium (MEM) can beemployed.

(c) Preparation of Myeloma Cells (Hereinafter Referred to as “Myeloma”)

The myeloma cells to be used for cell fusion are not particularlylimited and suitable cells can be selected from known cell lines.However, in consideration of convenience when a hybridoma is selectedfrom fused cells, it is preferred to use an HGPRT (hypoxanthine-guaninephosphoribosyl transferase) defective strain whose selection procedurehas been established.

More specifically, examples of the HGPRT defective strain includeX63-Ag8(X63), NS1-ANS/1(NS1), P3X63-Ag8.U1(P3U1), X63-Ag8.653(X63.653),SP2/0-Ag14(SP2/0), MPC11-45.6TG1.7(45.6TG), FO, S149/5XXO and BU.1derived from mice, 210.RSY3.Ag.1.2.3(Y3) derived from rats; andU266AR(SKO-007), GM1500.GTG-A12(GM1500), UC729-6, LICR-LOW-HMy2(HMy2)and 8226AR/NIP4-1(NP41) derived from humans.

These HGPRT defective strains are available from, for example, theAmerican Type Culture Collection (ATCC) or the like.

These cell strains are subcultured in an appropriate medium such as an8-azaguanine medium [a medium obtained by adding 8-azaguanine to anRPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol,gentamicin and fetal calf serum (hereinafter referred to as “FCS”)];Iscove's Modified Dulbecco's Medium (hereinafter referred to as “IMDM”),or Dulbecco's Modified Eagle Medium (hereinafter referred to as “DMEM”).In this case, 3 to 4 days before performing cell fusion, the cells aresubcultured in a normal medium [for example, an ASF104 medium(manufactured by Ajinomoto Co., Ltd.) containing 10% FCS] to obtain notless than 2×10⁷ cells on the day of cell fusion.

(d) Cell Fusion

Fusion between the antibody-producing cells and the myeloma cells isappropriately performed according to a known method (Weir, D. M.Handbook of Experimental Immunology Vol. I. II. III., BlackwellScientific Publications, Oxford (1987), Kabat, E. A. and Mayer, M. M.,Experimental Immunochemistry, Charles C Thomas Publisher, Springfield,Ill. (1964), etc.), under conditions such that the survival rate ofcells is not excessively reduced.

Examples of the method include a chemical method in which theantibody-producing cells and the myeloma cells are mixed in a highconcentration polymer solution of polyethylene glycol or the like, and aphysical method using electric stimulation.

Among these methods, a specific example of the chemical method is asdescribed below.

That is, in the case where polyethylene glycol is used for the highconcentration polymer solution, the antibody-producing cells and themyeloma cells are mixed in a solution of polyethylene glycol having amolecular weight of 1500 to 6000, more preferably 2000 to 4000 at atemperature of 30 to 40° C., preferably 35 to 38° C. for 1 to 10minutes, preferably 5 to 8 minutes.

(e) Selection of a Group of Hybridomas

The method of selecting hybridomas obtained by the above-mentioned cellfusion is not particularly limited. Usually, an HAT (hypoxanthine,aminopterin, thymidine) selection method (Kohler et al., Nature (1975),256, p. 495; Milstein et al., Nature (1977), 266, p. 550) is used.

This method is effective when hybridomas are obtained using the myelomacells of an HGPRT defective strain incapable of surviving in thepresence of aminopterin.

That is, by culturing unfused cells and hybridomas in an HAT medium,only hybridomas resistant to aminopterin are selectively allowed tosurvive and proliferate.

(f) Division into Single Cell Clone (Cloning)

As a cloning method for hybridomas, a known method such as amethylcellulose method, a soft agarose method, or a limiting dilutionmethod can be used (see, for example, Barbara, B. M. and Stanley, M. S.:Selected Methods in Cellular Immunology, W. H. Freeman and Company, SanFrancisco (1980)). Among these methods, particularly, a limitingdilution method is preferred.

In this method, a fibroblast cell strain derived from a rat fetus orfeeder cells such as normal mouse spleen cells, thymus gland cells, orascites cells are seeded in a microplate.

Meanwhile, hybridomas are diluted in a medium to give a cell density of0.2 to 0.5 cells per 0.2 ml. A 0.1 ml aliquot of the diluted hybridomasuspension is placed in each well and culturing is continued for about 2weeks while replacing about ⅓ of the culture solution with a freshmedium at predetermined time intervals (for example, every 3 days),whereby hybridoma clones can be proliferated.

The hybridomas in wells for which the antibody titer has been confirmedare subjected to, for example, cloning by the limiting dilution methodrepeatedly 2 to 4 times. A hybridoma which has been confirmed to have astable antibody titer is selected as an anti-Siglec-15 monoclonalantibody-producing hybridoma strain.

Examples of the hybridoma strain thus cloned include hybridoma #32A1 andhybridoma #41B1. Hybridoma #32A1 and hybridoma #41B1 were deposited atthe International Patent Organism Depositary of the National Instituteof Advanced Industrial Science and Technology (located at Central 6,1-1-1 Higashi, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Aug. 28,2008. The hybridoma #32A1 has been given a deposition number of FERMBP-10999 under the name of anti-Siglec-15 Hybridoma #32A1, and thehybridoma #41B1 has been given a deposition number of FERM BP-11000under the name of anti-Siglec-15 Hybridoma #41B1. In this description,the antibody produced by the hybridoma #32A1 is represented by “#32A1antibody” or simply “#32A1”, and the antibody produced by the hybridoma#41B1 is represented by “#41B1 antibody” or simply “#41B1”. Further,antibodies obtained in the Examples of this description other than fromhybridoma #32A1 and hybridoma #41B1 are also represented by the antibodynames in the same manner.

(g) Preparation of Monoclonal Antibody by Culturing Hybridoma

By culturing the thus selected hybridoma, a monoclonal antibody can beefficiently obtained. However, prior to the culturing, it is preferredto perform screening of a hybridoma which produces a target monoclonalantibody.

In the screening, a known method can be employed.

The measurement of the antibody titer in the invention can be carriedout by, for example, an ELISA method explained in item (b) describedabove.

The hybridoma obtained by the method as described above can be stored ina frozen state in liquid nitrogen or in a freezer at −80° C. or below.

After completion of cloning, the medium is changed from an HT medium toa normal medium, and the hybridoma is cultured.

Large-scale culture is performed by rotation culture using a largeculture bottle or by spinner culture.

From the supernatant obtained by the large-scale culture, a monoclonalantibody which specifically binds to the protein of the invention can beobtained by purification using a method known to those skilled in theart such as gel filtration.

Further, the hybridoma is injected into the abdominal cavity of a mouseof the same strain as the hybridoma (for example, the above-mentionedBALB/c) or a Nu/Nu mouse to proliferate the hybridoma, whereby theascites containing a large amount of the monoclonal antibody of theinvention can be obtained.

In the case where the hybridoma is administered in the abdominal cavity,if a mineral oil such as 2,6,10,14-tetramethyl pentadecane (pristine) isadministered 3 to 7 days prior thereto, a larger amount of the ascitescan be obtained.

For example, an immunosuppressant is previously injected into theabdominal cavity of a mouse of the same strain as the hybridoma toinactivate T cells. 20 days thereafter, 10⁶ to 10⁷ hybridoma clone cellsare suspended in a serum-free medium (0.5 ml), and the suspension isinjected into the abdominal cavity of the mouse. In general, when theabdomen is expanded and filled with the ascites, the ascites iscollected.

By this method, the monoclonal antibody can be obtained at aconcentration about 100-fold higher than that of the culture solution.

The monoclonal antibody obtained by the above-mentioned method can bepurified by the method described in, for example, Weir, D. M.: Handbookof Experimental Immunology Vol. I, II, III, Blackwell ScientificPublications, Oxford (1978).

That is, examples of the method include an ammonium sulfateprecipitation method, gel filtration, ion exchange chromatography, andaffinity chromatography.

As a simple purification method, a commercially available monoclonalantibody purification kit (for example, MAbTrap GII kit manufactured byPharmacia, Inc.) or the like can also be used.

The thus obtained monoclonal antibody has high antigen specificity forSiglec-15.

(h) Assay of Monoclonal Antibody

The isotype and subclass of the thus obtained monoclonal antibody can bedetermined as follows.

First, examples of the identification method include the Ouchterlonymethod, an ELISA method and an RIA method.

The Ouchterlony method is simple, but when the concentration of themonoclonal antibody is low, a condensation operation is required.

On the other hand, when an ELISA method or an RIA method is used, bydirectly reacting the culture supernatant with an antigen-adsorbed solidphase and using antibodies corresponding to various types ofimmunoglobulin isotypes and subclasses as secondary antibodies, theisotype and subclass of the monoclonal antibody can be identified.

In addition, as a simpler method, a commercially availableidentification kit (for example, Mouse Typer kit manufactured by Bio-RadLaboratories, Inc.) or the like can also be used.

Further, quantitative determination of a protein can be performed by theFolin Lowry method and a method of calculation based on the absorbanceat 280 nm [1.4 (OD 280)=Immunoglobulin 1 mg/ml].

(3) Other Antibodies

The antibody of the invention includes not only the above-mentionedmonoclonal antibody against Siglec-15 but also a recombinant antibodyobtained by artificial modification for the purpose of decreasingheterologous antigenicity to humans such as a chimeric antibody, ahumanized antibody and a human antibody. These antibodies can beproduced using a known method.

As the chimeric antibody, an antibody in which antibody variable andconstant regions are derived from different species, for example, achimeric antibody in which a mouse-derived antibody variable region isconnected to a human-derived constant region can be exemplified (seeProc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

As the humanized antibody, an antibody obtained by integrating only acomplementarity determining region (CDR) into a human-derived antibody(see Nature (1986) 321, pp. 522-525), and an antibody obtained bygrafting a part of the amino acid residues of the framework as well asthe CDR sequence into a human antibody by a CDR-grafting method (WO90/07861) can be exemplified.

Further, the antibody of the invention includes a human antibody. Ananti-Siglec-15 human antibody refers to a human antibody having only agene sequence of an antibody derived from a human chromosome. Theanti-Siglec-15 human antibody can be obtained by a method using a humanantibody-producing mouse having a human chromosome fragment containingH-chain and L-chain genes of a human antibody (see Tomizuka, K. et al.,Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nuc. AcidsRes. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal CellTechnology: Basic and Applied Aspects vol. 10, pp. 69-73 (Kitagawa, Y.,Matuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999;Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727,etc.).

Such a transgenic animal can be created specifically as follows. Agenetically modified animal in which non-human mammalian endogenousimmunoglobulin heavy and light chain gene loci have been disrupted, andinstead, human immunoglobulin heavy and light chain gene loci have beenintroduced via a yeast artificial chromosome (YAC) vector or the like iscreated by producing a knockout animal and a transgenic animal andmating these animals.

Further, according to a genetic engineering technique, by using cDNAsencoding such a heavy chain and a light chain of a human antibody,respectively, preferably a vector containing the cDNAs, eukaryotic cellsare transformed, and a transformant which produces a recombinant humanmonoclonal antibody is cultured, whereby the antibody can also beobtained from the culture supernatant.

Here, as the host, for example, eukaryotic cells, preferably mammaliancells such as CHO cells, lymphocytes or myeloma cells can be used.

Further, a method of obtaining a phage display-derived human antibodyscreened from a human antibody library (see Wormstone, I. M. et al.,Investigative Ophthalmology & Visual Science. (2002) 43 (7), pp.2301-2308; Carmen, S. et al., Briefings in Functional Genomics andProteomics (2002), 1 (2), pp. 189-203; Siriwardena, D. et al.,Opthalmology (2002) 109 (3), pp. 427-431, etc.) is also known.

For example, a phage display method in which a variable region of ahuman antibody is expressed on the surface of a phage as a single-chainantibody (scFv), and a phage which binds to an antigen is selected(Nature Biotechnology (2005), 23, (9), pp. 1105-1116) can be used.

By analyzing the gene of the phage selected based on the binding to anantigen, the DNA sequence encoding the variable region of a humanantibody which binds to an antigen can be determined.

If the DNA sequence of scFv which binds to an antigen is determined, ahuman antibody can be obtained by preparing an expression vector havingthe sequence and introducing the vector into an appropriate host toexpress it (WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO93/19172, WO 95/01438, WO 95/15388, Annu Rev. Immunol (1994) 12, pp.433-455, Nature Biotechnology (2005) 23 (9), pp. 1105-1116).

In the case where an antibody is produced by once isolating an antibodygene and then introducing the gene into an appropriate host, acombination of appropriate host and expression vector can be used.

In the case where eukaryotic cells are used as the host, animal cells,plant cells and eukaryotic microorganisms can be used.

As the animal cells, mammalian cells, for example, dihydrofolatereductase-deficient strains (Urlaub, G. and Chasin, L. A., Proc. Natl.Acad. Sci. USA (1980) 77, pp. 4126-4220) of simian COS cells (Gluzman,Y., Cell, (1981) 23, pp. 175-182, ATCC CRL-1650), murine fibroblastsNIH3T3 (ATCC No. CRL-1658), and Chinese hamster ovarian cells (CHOcells; ATCC: CCL-61); can be exemplified.

In cases where prokaryotic cells are used, for example, Escherichia coliand Bacillus subtilis can be exemplified.

By introducing a gene of a target antibody into these cells throughtransformation, and culturing the thus transformed cells in vitro, theantibody can be obtained.

There is no limitation on isotype of the antibody of the invention, andexamples thereof include IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1,IgA2), IgD and IgE, and preferred examples thereof include IgG and IgM.

Further, the antibody of the invention may be a functional fragment ofthe antibody having an antigen-binding site of the antibody or amodified fragment thereof. The fragment of the antibody can be obtainedby treating the antibody with a protease such as papain or pepsin, ormodifying the antibody gene according to a genetic engineering techniqueand expressing the modified gene in suitable cultured cells. Among theseantibody fragments, a fragment having all or part of the functions ofthe full-length molecule of the antibody can be called a functionalfragment of the antibody. As the functions of the antibody, generally anantigen-binding activity, an activity of neutralizing the activity of anantigen, an activity of increasing the activity of an antigen, anantibody-dependent cytotoxic activity, a complement-dependent cytotoxicactivity, and a complement-dependent cellular cytotoxic activity can beexemplified. The function of the functional fragment of the antibodyaccording to the invention is preferably an activity of inhibiting theformation of osteoclasts, more preferably an activity of inhibiting theprocess of cell fusion of osteoclasts.

Examples of the fragment of the antibody include Fab, F(ab′)2, Fv,single-chain Fv (scFv) in which Fv molecules of the heavy chain and thelight chain are ligated via an appropriate linker, a diabody(diabodies), a linear antibody, and a polyspecific antibody composed ofthe antibody fragment. Further, Fab′ which is a monovalent fragment in avariable region of an antibody obtained by treating F(ab′)2 underreducing conditions is also included in the fragment of the antibody.

Further, the antibody of the invention may be a polyspecific antibodywith specificity for at least two different antigens.

In general, such a molecule binds to two antigens (that is, bispecificantibody), however, the “polyspecific antibody” as used herein includesan antibody having specificity for two or more (for example, three)antigens.

The antibody of the invention may be a polyspecific antibody composed ofa full-length antibody or a fragment of such an antibody (for example,F(ab′)2 bispecific antibody). The bispecific antibody can be produced byligating the heavy and light chains (HL pairs) of two types ofantibodies, or can also be produced by fusing hybridomas which producedifferent monoclonal antibodies to prepare fused cells which produce thebispecific antibody (Millstein et al., Nature (1983) 305, pp. 537-539).

The antibody of the invention may be a single-chain antibody (alsoreferred to as scFv). The single-chain antibody can be obtained byligating the V regions of the heavy chain and the light chain of theantibody via a polypeptide linker (Pluckthun, The Pharmacology ofMonoclonal Antibodies, 113 (edited by Rosenburg and Moore, SpringerVerlag, New York, pp. 269-315 (1994), Nature Biotechnology (2005), 23,pp. 1126-1136). Further, a BiscFv fragment produced by ligating two scFvmolecules via a polypeptide linker can also be used as the bispecificantibody.

A method of producing a single-chain antibody is known in this technicalfield (see, for example, U.S. Pat. Nos. 4,946,778, 5,260,203, 5,091,513,5,455,030, etc.). In this scFv, the V regions of the heavy chain and thelight chain are ligated via a linker which does not form a conjugate,preferably via a polypeptide linker (Huston, J. S. et al., Proc. Natl.Acad. Sci. USA (1988), 85, pp. 5879-5883). In the scFv, the V regions ofthe heavy chain and the light chain may be derived from the sameantibody or different antibodies. As the polypeptide linker to be usedfor ligating the V regions, for example, a given single-chain peptidecomposed of 12 to 19 residues is used.

DNA encoding scFv can be obtained by performing amplification using apartial DNA encoding the entire or a desired amino acid sequence of aDNA selected from a DNA encoding the heavy chain or the V region of theheavy chain of the above-mentioned antibody and a DNA encoding the lightchain or the V region of the light chain thereof as a template by a PCRmethod using a primer pair that defines both ends thereof, and furtherperforming amplification combining a DNA encoding a polypeptide linkerportion and a primer pair that defines both ends thereof so as to ligateboth the ends to the heavy chain and the light chain, respectively.

Further, once DNA encoding scFv is produced, an expression vectorcontaining the same and a host transformed by the expression vector canbe obtained according to a common procedure. Further, by using theresulting host, scFv can be obtained according to a common procedure.

An antibody fragment thereof can be produced in a host by obtaining agene and expressing the gene in the same manner as described above.

The antibody of the invention may be multimerized to increase theaffinity for an antigen. The antibody to be multimerized may be one typeof antibody or a plurality of antibodies which recognize a plurality ofepitopes of the same antigen. As a method of multimerization of theantibody, binding of the IgG CH3 domain to two scFv molecules, bindingto streptavidin, introduction of a helix-turn-helix motif and the likecan be exemplified.

The antibody of the invention may be a polyclonal antibody which is amixture of plural types of anti-Siglec-15 antibodies having differentamino acid sequences. As one example of the polyclonal antibody, amixture of plural types of antibodies having different CDR can beexemplified. As such a polyclonal antibody, a mixture of cells whichproduce different antibodies is cultured, and an antibody purified fromthe resulting culture can be used (see WO 2004/061104).

As a modified antibody, an antibody bound to any of various types ofmolecules such as polyethylene glycol (PEG) can also be used.

Further, the antibody of the invention may be in the form of a conjugateformed between any of these antibodies and another medicinal agent(immunoconjugate). Examples of such an antibody include one in which theantibody is conjugated to a radioactive material or a compound having apharmacological action (Nature Biotechnology (2005) 23, pp. 1137-1146).

The obtained antibody can be purified to homogeneity. The separation andpurification of the antibody can be performed employing a conventionalprotein separation and purification method.

For example, the antibody can be separated and purified by appropriatelyselecting and combining use of a chromatography column, filter,ultrafiltration, salt precipitation, dialysis, preparativepolyacrylamide gel electrophoresis, isoelectric focusingelectrophoresis, and the like (Strategies for Protein Purification andCharacterization: A Laboratory Course Manual, Daniel R. Marshak et al.eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: ALaboratory Manual. Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988)), but the method is not limited thereto.

Examples of chromatography include affinity chromatography, ion exchangechromatography, hydrophobic chromatography, gel filtrationchromatography, reverse phase chromatography, and adsorptionchromatography.

Such chromatography can be performed employing liquid chromatographysuch as HPLC or FPLC.

As a column to be used in affinity chromatography, a Protein A columnand a Protein G column can be exemplified.

For example, as a column using a Protein A column, Hyper D, POROS,Sepharose F. F. (Pharmacia) and the like can be exemplified.

Further, by using a carrier having an antigen immobilized thereon, theantibody can also be purified utilizing the binding property of theantibody to the antigen.

5. Medicine Containing Anti-Siglec-15 Antibody

From the anti-Siglec-15 antibodies obtained by the method described inthe above item “4. Production of anti-Siglec-15 antibody”, an antibodywhich neutralizes the biological activity of Siglec-15 can be obtained.Such an antibody which neutralizes the biological activity of Siglec-15inhibits the biological activity of Siglec-15 in vivo, i.e., thedifferentiation and/or maturation of osteoclasts, and therefore can beused as a therapeutic and/or preventive agent for abnormal bonemetabolism caused by abnormal differentiation and/or maturation ofosteoclasts as a medicine. The abnormal bone metabolism may be anydisorder characterized by net bone loss (osteopenia or osteolysis). Ingeneral, the treatment and/or prevention by the anti-Siglec-15 antibodyare/is applied to a case where inhibition of bone resorption isrequired. Examples of the abnormal bone metabolism which can be treatedand/or prevented by the anti-Siglec-15 antibody include osteoporosis(postmenopausal osteoporosis, senile osteoporosis, secondaryosteoporosis due to the use of a therapeutic agent such as a steroid oran immunosuppressant, or osteoporosis accompanying rheumatoidarthritis), bone destruction accompanying rheumatoid arthritis,cancerous hypercalcemia, bone destruction accompanying multiple myelomaor cancer metastasis to bone, giant cell tumor, tooth loss due toperiodontitis, osteolysis around a prosthetic joint, bone destruction inchronic osteomyelitis, Paget's disease of bone, renal osteodystrophy andosteogenesis imperfecta, however, the abnormal bone metabolism is notlimited thereto as long as it is a disease accompanied by net bone losscaused by osteoclasts. Examples of the anti-Siglec-15 antibody to beused as the above-mentioned medicine include a chimeric antibody and ahumanized antibody produced from the #32A1 antibody or #41B1 antibody bythe method described in 4. (3) “Other antibodies”. Further, a chimericantibody, a humanized antibody and a human antibody sharing the sameepitope as the #32A1 antibody or #41B1 antibody can also be used as amedicine. Whether a certain anti-Siglec-15 antibody shares the sameepitope as the #32A1 antibody or #41B1 antibody can be confirmed byobserving whether or not these antibodies bind to the same specificpartial peptide of Siglec-15. Further, it can also be determined that ifthe certain anti-Siglec-15 antibody competes with the #32A1 antibody or#41B1 antibody for binding to Siglec-15, these antibodies share the sameepitope.

The in vitro activity of the anti-Siglec-15 antibody of neutralizing thebiological activity of Siglec-15 can be determined by, for example, theactivity of inhibiting the differentiation of the cells whichoverexpress Siglec-15 into osteoclasts. For example, the anti-Siglec-15antibody is added to RAW 264.7 cells or Raw 264 cells which are a mousemonocyte-derived cell line at various concentrations, and the activityof inhibiting the differentiation into osteoclasts by stimulation withRANKL or TNF-α can be determined. Further, the anti-Siglec-15 antibodyis added to bone marrow-derived primary cultured cells at variousconcentrations, and the activity of inhibiting the differentiation intoosteoclasts by stimulation with RANKL, TNF-α or active vitamin D₃ can bedetermined. Further, the anti-Siglec-15 antibody is added to normalhuman osteoclast precursor cells (Normal Human Natural OsteoclastPrecursor Cells, available from Sanko Junyaku Co., Ltd., Cat. No.2T-110) at various concentrations, and the activity of inhibiting thedifferentiation into osteoclasts by stimulation with RANKL or M-CSF canbe determined. Such an inhibitory effect on osteoclast differentiationcan be determined by using the inhibition of tartrate-resistant acidphosphatase (TRAP) activity of osteoclasts as an index as shown inExamples 17, 19, 20 and 26. Further, the inhibitory effect on osteoclastdifferentiation can also be determined by using the inhibition offormation of TRAP-positive multinucleated osteoclasts, i.e., theinhibition of cell fusion of osteoclasts as an index as shown inExamples 19, 21, 22 and 35. The antibodies of the invention exhibited aninhibitory effect on cell fusion at a concentration of 30 μg/ml or lessin the above-mentioned test system for osteoclast differentiation, andsome antibodies exhibited the inhibitory effect even at a concentrationof 3 μg/ml or less or 1 μg/ml or less. Further, in the case where theeffect at a further lower concentration was tested, it was found that aplurality of antibodies exhibited an inhibitory effect on osteoclastdifferentiation even at a concentration range of from 63 ng/ml to 1μg/ml. Further, in an experiment of a pit assay (Takada et al., Bone andMineral, (1992) 17, 347-359) using femur- and/or tibia-derived cells,the in vitro activity of inhibiting the bone resorption by osteoclastscan be determined by adding the anti-Siglec-15 antibody to femur- and/ortibia-derived cells at various concentrations, and observing pitformation on a dentine slice. As a system for determining the in vitroactivity of inhibiting the bone resorption by osteoclasts, as shown inExample 37, it is also possible to use a plate coated with humancollagen conjugated to europium. In the above-mentioned test system forbone resorption by osteoclasts, the antibody of the invention inhibitedbone resorption at a concentration of 3 μg/ml or less, that is, at aconcentration range of from 0.3 μg/ml to 3 μg/ml. The in vivotherapeutic or preventive effect of the anti-Siglec-15 antibody onabnormal bone metabolism using an experimental animal can be confirmedby administering the anti-Siglec-15 antibody to a model animal ofosteoporosis or a transgenic animal which overexpresses siglec-15 andmeasuring a change in osteoclasts.

The thus obtained antibody which neutralizes the biological activity ofSiglec-15 is useful as a medicine, particularly as a pharmaceuticalcomposition for treating or preventing abnormal bone metabolism such asosteoporosis, bone destruction accompanying rheumatoid arthritis or bonedestruction accompanying cancer metastasis to bone, or as an antibodyfor immunological diagnosis of such a disease.

In the treatment of rheumatoid arthritis (RA), a major problem is boneloss accompanying the occurrence of the disease. It has been reportedthat in this bone loss accompanying RA, osteoclasts play a primary role.The cytokines considered to be most important for osteoclast induction(differentiation and maturation) and activation and the cause of bonedestruction in RA are RANKL and TNF-α (Romas E. et al., Bone 30, pp.340-346, 2002). As shown in Example 19 of this description, OCIF/OPGwhich is a decoy receptor for RANKL can inhibit osteoclast formationinduced by RANKL but does not inhibit osteoclast formation induced byTNF-α. On the other hand, the anti-Siglec-15 antibody according to theinvention effectively inhibited osteoclast formation induced by bothRANKL and TNF-α. Therefore, it is expected that the anti-Siglec-15antibody of the invention can inhibit bone loss and bone destructioninduced by TNF-α in RA or the like more strongly than an RANKL blocker(OCIF/OPG, an anti-RANKL antibody or the like).

As one example, for the treatment or prevention of abnormal bonemetabolism, the anti-Siglec-15 antibody can be administered alone oralong with at least one therapeutic agent for a bone-related disease. Asanother example, the anti-Siglec-15 antibody can be administered alongwith a therapeutically effective amount of a therapeutic agent forabnormal bone metabolism. Examples of the therapeutic agent which can beadministered along with the anti-Siglec-15 antibody include, but are notlimited to, bisphosphonates, active vitamin D₃, calcitonin andderivatives thereof, hormone preparations such as estradiol, SERMs(selective estrogen receptor modulators), ipriflavone, vitamin K₂(menatetrenone), calcium preparations, PTH (parathyroid hormone)preparations, nonsteroidal anti-inflammatory agents, soluble TNFreceptor preparations, anti-TNF-α antibodies or functional fragments ofthe antibodies, anti-PTHrP (parathyroid hormone-related protein)antibodies or functional fragments of the antibodies, IL-1 receptorantagonists, anti-IL-6 receptor antibodies or functional fragments ofthe antibodies, anti-RANKL antibodies or functional fragments of theantibodies and OCIF (osteoclastogenesis inhibitory factor). Depending onthe state of abnormal bone metabolism or the intended degree of thetreatment and/or prevention, two or three, or more types of medicinalagents can be administered, and these medicinal agents can be suppliedall together by encapsulating them in the same preparation. Thesemedicinal agents and the anti-Siglec-15 antibody can be supplied alltogether by encapsulating them in the same preparation. Further, thesemedicinal agents can be supplied all together by encapsulating them as akit to be used for treatment and/or prevention. Further, these medicinalagents and the anti-Siglec-15 antibody can be supplied separately. Inthe case of administration in gene therapy, a gene of a proteinoustherapeutic agent for a bone disease and a gene of the anti-Siglec-15antibody can be inserted downstream of the same promoter region ordifferent promoter regions, and can be introduced into the same vectoror different vectors.

By conjugating a therapeutic agent for a bone disease to theanti-Siglec-15 antibody or a fragment thereof, a targeted drug conjugateas described in M. C. Garnet “Targeted drug conjugates: principles andprogress”, Advanced Drug Delivery Reviews, (2001) 53, 171-216 can beproduced. For achieving this purpose, other than the antibody molecule,any antibody fragment can be applied as long as it does not completelylose the ability to recognize osteoclasts, and examples thereof includefragments such as Fab, F(ab′)2, and Fv. In the invention, the antibodyand the fragment can be used in the same manner. The conjugate formed bythe anti-Siglec-15 antibody or a fragment thereof and a therapeuticagent for a bone disease can be any of various forms described in M. C.Garnet “Targeted drug conjugates: principles and progress”, AdvancedDrug Delivery Reviews, (2001) 53, 171-216, G. T. Hermanson “BioconjugateTechniques” Academic Press, California (1996), Putnam and J. Kopecek“Polymer Conjugates with Anticancer Activity” Advances in PolymerScience (1995) 122, 55-123 and the like. That is, a conjugate form inwhich the anti-Siglec-15 antibody and a therapeutic agent for a bonedisease are conjugated to each other chemically and directly or via aspacer such as an oligopeptide and a conjugate form via an appropriatedrug carrier can be exemplified. Examples of the drug carrier include aliposome and a water-soluble polymer. More specific examples of theconjugate form via such a drug carrier include a conjugate form in whichthe antibody and a therapeutic agent for a bone disease are incorporatedin a liposome and the liposome and the antibody are conjugated to eachother, and a conjugate form in which a therapeutic agent for a bonedisease is conjugated to a water-soluble polymer (a compound having amolecular weight of from about 1000 to 100000) chemically and directlyor via a spacer such as an oligopeptide and the antibody is conjugatedto the water-soluble polymer. The conjugation of the antibody (or afragment thereof) to a therapeutic agent for a bone disease or a drugcarrier such as a liposome or a water-soluble polymer can be effected bya method known to those skilled in the art such as the method describedin G. T. Hermanson “Bioconjugate Techniques” Academic Press, California(1996), Putnam and J. Kopecek “Polymer Conjugates with AnticancerActivity” Advances in Polymer Science (1995) 122, 55-123. Theincorporation of a therapeutic agent for a bone disease in a liposomecan be effected by a method known to those skilled in the art such asthe method described in D. D. Lasic “Liposomes: From Physics toApplications” Elsevier Science Publishers B. V., Amsterdam (1993) or thelike. The conjugation of a therapeutic agent for a bone disease to awater-soluble polymer can be effected by a method known to those skilledin the art such as the method described in D. Putnam and J. Kopecek“Polymer Conjugates with Anticancer Activity” Advances in PolymerScience (1995) 122, 55-123. A conjugate between the antibody (or afragment thereof) and a proteinous therapeutic agent for a bone disease(or a fragment thereof) can be produced by a method known to thoseskilled in the art through genetic engineering other than theabove-mentioned method.

The invention also provides a pharmaceutical composition containing atherapeutically and/or preventively effective amount of theanti-Siglec-15 antibody and a pharmaceutically acceptable diluent,carrier, solubilizing agent, emulsifying agent, preservative and/oradjuvant.

The invention also provides a pharmaceutical composition containing atherapeutically and/or preventively effective amount of theanti-Siglec-15 antibody, a therapeutically and/or preventively effectiveamount of at least one therapeutic agent for a bone disease, and apharmaceutically acceptable diluent, carrier, solubilizing agent,emulsifying agent, preservative and/or adjuvant. Examples of thetherapeutic agent for a bone disease include, but are not limited to,bisphosphonates, active vitamin D₃, calcitonin and derivatives thereof,hormone preparations such as estradiol, SERMs (selective estrogenreceptor modulators), ipriflavone, vitamin K₂ (menatetrenone), calciumpreparations, PTH (parathyroid hormone) preparations, nonsteroidalanti-inflammatory agents, soluble TNF receptor preparations, anti-TNF-αantibodies or functional fragments of the antibodies, anti-PTHrP(parathyroid hormone-related protein) antibodies or functional fragmentsof the antibodies, IL-1 receptor antagonists, anti-IL-6 receptorantibodies or functional fragments of the antibodies, anti-RANKLantibodies or functional fragments of the antibodies and OCIF(osteoclastogenesis inhibitory factor).

A substance to be used in a preparation acceptable in a pharmaceuticalcomposition according to the present invention is preferably non-toxicto a person to which the pharmaceutical composition is to beadministered, in terms of the dose and concentration. The pharmaceuticalcomposition of the invention can contain a substance for pharmaceuticaluse which is capable of changing or maintaining the pH, osmoticpressure, viscosity, transparency, color, isotonicity, color, asepticcondition, stability, solubility, release rate, absorption rate, andpermeability. Examples of the substance for pharmaceutical use include,but are not limited to, amino acids such as glycine, alanine, glutamine,asparagine, arginine and lysine; antimicrobial agents; antioxidants suchas ascorbic acid, sodium sulfate and sodium hydrogen sulfite; bufferssuch as phosphate, citrate, borate buffers, bicarbonate and Tris-HClsolutions; fillers such as mannitol and glycine; chelating agents suchas ethylenediamine tetraacetate (EDTA); complexing agents such ascaffeine, polyvinylpyrrolidine, β-cyclodextrin andhydroxypropyl-β-cyclodextrin; expanders such as glucose, mannose anddextrin; other carbohydrates such as monosaccharides and disaccharides;coloring agents; flavors; diluents; emulsifying agents; hydrophilicpolymers such as polyvinylpyrrolidine; preservatives such as lowmolecular weight polypeptides, base forming counter ions, benzalkoniumchloride, benzoate, salicylic acid, thimerosal, phenethyl alcohol,methylparaben, propylparaben, chlorhexidine, sorbic acid, and hydrogenperoxide; solvents such as glycerin, propylene glycol and polyethyleneglycol; sugar alcohols such as mannitol and sorbitol; suspending agents;surfactants such as sorbitan ester, polysorbates including polysorbate20 and polysorbate 80, Triton, tromethamine, lecithin and cholesterol;stability enhancing agents such as sucrose and sorbitol; elasticityenhancing agents such as sodium chloride, potassium chloride andmannitol and sorbitol; transport agents; diluents; excipients; and/orpharmaceutical adjuvants. The addition amount of these substances forpharmaceutical use is preferably from 0.01 to 100 times, particularlypreferably from 0.1 to 10 times the weight of the anti-Siglec-15antibody. Those skilled in the art can appropriately determine apreferred formulation of the pharmaceutical composition in a preparationdepending on the disease to be applied, the route of administration tobe applied or the like.

The excipient or carrier in the pharmaceutical composition may be in theform of a liquid or a solid. An appropriate excipient or carrier may beinjectable water, physiological saline, an artificial cerebral spinalfluid or other substance commonly used for parenteral administration.Further, neutral physiological saline or physiological saline containingserum albumin can also be used as a carrier. The pharmaceuticalcomposition may contain a Tris buffer of pH 7.0 to 8.5 or an acetatebuffer of pH 4.0 to 5.5 which may be supplemented with sorbitol oranother compound. Examples of the pharmaceutical composition of theinvention include a pharmaceutical composition containing theanti-Siglec-15 antibody and a pharmaceutical composition containing theanti-Siglec-15 antibody and at least one therapeutic agent for a bonedisease. The pharmaceutical composition of the invention is prepared inthe form of a lyophilized product or a liquid as a medicinal agenthaving a selected composition and a required purity. The pharmaceuticalcomposition containing the anti-Siglec-15 antibody and thepharmaceutical composition containing the anti-Siglec-15 antibody and atleast one therapeutic agent for abnormal bone metabolism can also beformed into a lyophilized product using an appropriate excipient such assucrose.

The pharmaceutical composition of the invention can be prepared forparenteral administration or for gastrointestinal absorption throughoral administration. The composition and concentration of a preparationcan be determined depending on the administration method. As theaffinity of the anti-Siglec-15 antibody contained in the pharmaceuticalcomposition of the invention for Siglec-15 is higher, that is, as thedissociation constant (Kd value) for Siglec-15 is lower, theanti-Siglec-15 antibody can exhibit its drug efficacy at a lower dosefor humans, and therefore, the dose of the pharmaceutical composition ofthe invention for humans can also be determined based on this result. Asfor the dose, in the case where a human anti-Siglec-15 antibody isadministered to humans, the antibody may be administered at a dose offrom about 0.1 to 100 mg/kg once per one to 180 days.

Examples of the dosage form of the pharmaceutical composition of theinvention include injections including infusions, suppositories,transnasal agents, sublingual agents and percutaneous absorbents.

6. Search for Directly Interacting Substance

Another embodiment of the invention includes a drug design method basedon the conformation of Siglec-15 for obtaining a substance whichinhibits the activity of Siglec-15. Such a method is known as a rationaldrug design method and is used for searching for a compound whichefficiently inhibits or activates the enzymatic activity or binding to aligand, a cofactor or a DNA. As an example of such a compound, aprotease inhibitor serving as an anti-HIV agent which has already beenplaced on the market is well known. Also in a three-dimensionalstructural analysis of Siglec-15 of the invention, a generally wellknown method such as an X-ray crystallographic analysis or a nuclearmagnetic resonance method can be used. Further, in searching for asubstance which inhibits the function of Siglec-15, drug designutilizing computer-aided drug design (CADD) can also be effected. As anexample of this case, a low molecular weight compound (WO 99/58515)which inhibits the action of AP-1 and is expected to be a novel genomicdrug for treating rheumatoid arthritis and the like are known. By virtueof such a method, it is possible to obtain a substance which inhibitsthe function of Siglec-15 by directly binding to Siglec-15 or byinhibiting the interaction between Siglec-15 and other factors.

Further, another embodiment relates to a polypeptide with which theSiglec-15 of the invention associates, in other words, a partner proteinof the Siglec-15. That is, the invention relates to a method ofscreening a partner protein which regulates the activity of Siglec-15.

One embodiment of this screening method includes a step of bringing atest protein sample into contact with Siglec-15 and selecting a proteinwhich binds to Siglec-15. As such a method, for example, a method inwhich purified Siglec-15 is used and affinity purification of a proteinbinding thereto is performed can be exemplified. One specific example ofthe method will be described below. A sequence composed of sixhistidines as an affinity tag is fused with Siglec-15 to prepare afusion protein, and the resulting protein is incubated at 4° C. for 12hours along with a cell extract solution (a fraction passed through anickel-agarose column after charging the column with a cell solution).Then, another nickel-agarose carrier is added to the mixture and themixture is incubated at 4° C. for 1 hour. After the nickel-agarosecarrier is sufficiently washed with a washing buffer, 100 mM imidazoleis added to the mixture to elute and purify a protein which specificallybinds to Siglec-15 in the cell extract solution. Then, the purifiedprotein is analyzed to determine its structure. In this manner, aprotein which directly binds to Siglec-15 and a protein which does nothave an activity of binding to Siglec-15 but indirectly binds toSiglec-15 by forming a complex with a protein as a subunit whichdirectly binds to Siglec-15 can be purified. As an alternative method,it is also possible to perform cloning by a far-Western blotting assayor a two-hybrid system assay using yeast or mammalian cells, however, itis not limited to these methods.

If a cDNA of a partner protein which directly or indirectly interactswith Siglec-15 is obtained in this manner, the cDNA can be used infunctional screening of a substance which inhibits the interactionbetween Siglec-15 and the partner protein. Specifically, a fusionprotein between Siglec-15 and glutathione-S-transferase is prepared andbound to a microplate covered with an anti-glutathione-S-transferaseantibody. Then, a biotinylated partner protein is brought into contactwith the fusion protein, and the binding of the partner protein with thefusion protein is detected using streptavidin-conjugated alkalinephosphatase. When the biotinylated partner protein is added, a testsubstance is also added to select a substance which promotes or inhibitsthe binding of the fusion protein to the partner protein. By thismethod, a substance which directly acts on the fusion protein or asubstance which directly acts on the partner protein can be obtained.

In the case where the fusion protein binds indirectly to the partnerprotein via another factor, the assay is performed in the presence of,for example, a cell extract solution containing this factor. In thiscase, there is a possibility that a substance which may act on thefactor may also be selected.

Further, in the case where the obtained partner protein has the activityof promoting the function of Siglec-15, it is possible to screen acandidate substance useful as a therapeutic and/or preventive agent forabnormal bone metabolism, for example, a therapeutic and/or preventiveagent for osteoporosis according to a test method employing anexpression vector of the Siglec-15 gene as described above. Further, inthe case where the obtained partner protein has an activity ofinhibiting the function of Siglec-15, it is possible to use apolynucleotide encoding such an inhibitor in gene therapy for abnormalbone metabolism.

Such a polynucleotide can be obtained by, for example, analyzing theamino acid sequence of the identified inhibitor, synthesizing anoligonucleotide probe encoding the amino acid sequence, and performingscreening of a cDNA library or a genome library. Further, in the casewhere a polypeptide having the activity of inhibiting the function ofSiglec-15 is derived from a randomly synthesized artificial peptidelibrary, a DNA composed of a nucleotide sequence encoding the amino acidsequence of the peptide is chemically synthesized.

In the gene therapy, the thus obtained gene encoding the inhibitor isintegrated into, for example, a viral vector and a patient is infectedwith a virus (attenuated) having the resultant recombinant viral vector.In the body of the patient, an anti-bone destruction factor is producedand functions to inhibit osteoclast differentiation, and therefore,treatment and/or prevention of abnormal bone metabolism can be realized.

As a method of introducing a gene therapeutic agent into a cell, eithera gene transfer method using a viral vector or a non-viral gene transfermethod can be used.

Examples of the gene transfer method using a viral vector include amethod of integrating a DNA encoding a Siglec-15 inhibitor or a mutantthereof into a DNA virus or an RNA virus such as a retrovirus, anadenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus,a pox virus, a polio virus, or a sindbis virus to effect gene transfer.Among them, a method using a retrovirus, an adenovirus, anadeno-associated virus, or a vaccinia virus is particularly preferred.Examples of the non-viral gene transfer method include a method ofadministering an expression plasmid directly into the muscle (a DNAvaccination method), a liposome method, a lipofection method, amicroinjection method, a calcium phosphate method, and anelectroporation method. In particular, a DNA vaccination method and aliposome method are preferred.

Further, in order to actually use the gene therapeutic agent as amedicinal agent, there are an in vivo method in which a DNA is directlyintroduced in the body and an ex vivo method in which a certain type ofcell is taken from a human and a DNA is introduced into the cell exvivo, and the cell is returned in the body.

For example, in the case where the gene therapeutic agent isadministered by the in vivo method, it is administered through anappropriate administration route such as through a vein or artery, underthe skin, into the skin, or into the muscle according to the disease,symptoms or the like. Further, in the case where it is administered bythe in vivo method, the gene therapeutic agent is generally formulatedas an injection, however, a commonly used carrier may be added asneeded. Further, in the case where it is formed into a liposome or amembrane fusion liposome (such as Sendai virus liposome), it can beformulated into a liposome preparation such as a suspension, alyophilized agent, or a centrifugally concentrated and lyophilizedagent.

A nucleotide sequence complementary to a full-length or partial sequenceof the nucleotide sequence represented by SEQ ID NO: 1 or 3 in theSequence Listing can be used for so-called antisense therapy. Anantisense molecule can be used as a DNA which is composed generally offrom 15 to 30 nucleotides and is complementary to a part of a nucleotidesequence selected from the nucleotide sequences represented by SEQ IDNOS: 1 and 3 in the Sequence Listing or a stable DNA derivative thereofsuch as a phosphorothioate, methylphosphonate, or morpholino derivativethereof, or a stable RNA derivative such as 2′-O-alkyl RNA. Such anantisense molecule can be introduced into a cell by a method known inthe technical field of the invention, for example, by injecting anextremely small amount of the antisense molecule, by forming themolecule into a liposome capsule, or by expressing the molecule with theuse of a vector having an antisense sequence. Such antisense therapy isuseful for treating a disease caused by excessively increasing theactivity of a protein encoded by the nucleotide sequence represented bySequence ID NO: 1 or 3 in the Sequence Listing.

Further, a method using a double-stranded short RNA (siRNA) can also beexemplified (Genes and Developments, Jan. 15, 2001, vol. 15, No. 2, pp.188-200). For example, siRNA against Siglec-15 gene is prepared andintroduced into a cell according to the method described in thedocument, whereby a therapeutic agent for a bone metabolic diseaseaccompanied by overexpression of Siglec-15 can be prepared.

EXAMPLES

Hereinafter, the invention will be more specifically described withreference to Examples, however, the invention is not limited thereto.Note that the respective operations regarding gene manipulation in thefollowing Examples were performed according to the methods described in“Molecular Cloning” (written by Sambrook, J., Fritsch, E. F. andManiatis, T., published by Cold Spring Harbor Laboratory Press in 1989),or in the case of using commercially available reagents or kits,performed according to the protocols attached thereto.

Example 1 Expression of Human Siglec-15 Gene in Giant Cell Tumor Tissue

A giant cell tumor (GCT) is histologically a bone tumor with a largenumber of osteoclast-like multinucleated giant cells arising and ischaracterized by clinical findings of osteolytic bone destruction(Bullough et al., Atlas of Orthopedic Pathology 2nd edition, 17.6-17.8,Lippincott Williams & Wilkins Publishers (1992)). An expression profileanalysis was performed for an EST probe (Affymetrix GeneChip HG-U133probe 215856_at: manufactured by Affymetrix, Inc.) having a nucleotidesequence partially overlapping with that of human Siglec-15 gene in GCTtissues using the database (Genesis 2006 Release 3.0) made by GeneLogic, Inc. Further, an expression profile analysis was also performedfor the EST probes for RANK (Affymetrix GeneChip HG-U133 probe207037_at, manufactured by Affymetrix, Inc.) and RANKL (AffymetrixGeneChip HG-U133 probe 210643_at, manufactured by Affymetrix, Inc.)which play a key role in differentiation into osteoclasts, and forcathepsin K (Affymetrix GeneChip HG-U133 probe 202450_s_at, manufacturedby Affymetrix, Inc.) and TRAP (Affymetrix GeneChip HG-U133 probe204638_at, manufactured by Affymetrix, Inc.) which are markers fordifferentiation into osteoclasts in GCT tissues in the same manner.

When the expression levels were compared among 13 cases of normal bonetissues, 12 cases of GCT tissues and 16 cases of bone tumor tissuesother than GCT, it was revealed that transcription of RANK and RANKL isspecifically increased in the GCT tissues compared with in the normaltissues (FIG. 1-A). On the other hand, in the bone tumor tissues otherthan GCT in which an increase in bone resorption is believed not to bealways caused, transcription of RANK and RANKL was lower than in GCT,and therefore, it was suggested that GCT provides an environment inwhich osteoclast formation and activation are promoted. Further, whenthe expression levels of genes of cathepsin K and TRAP were compared,the genes were transcribed at a high level in GCT (FIG. 1-B) and it wassuggested that a large number of osteoclasts having a bone resorptionactivity arise. Similarly, when the transcription levels of Siglec-15gene were compared, it was revealed that the gene was transcribed at ahigh level specifically in GCT in the same manner as the respectiveRANK, RANKL, cathepsin K and TRAP genes (FIG. 2). From these results, itwas suggested that Siglec-15 is associated with human pathology in whichbone resorption is increased as GCT.

Example 2 Extraction of Total RNA from Mouse-Derived Mature Osteoclasts

a) Mouse monocyte-derived cells RAW 264.7 (ATCC Cat. No. TIB-71) wereprepared at 4.5×10⁴ cells/ml in α-MEM medium containing 10% fetal bovineserum. The resulting cell preparation was put in a 75 cm² flask at 10ml/flask, and human RANKL (manufactured by PeproTech Inc.) was addedthereto to give a final concentration of 40 ng/ml, and the cells werecultured for 3 days in a CO₂ incubator. Further, the culturing withoutthe addition of human RANKL was performed in the same manner.

After completion of the culturing, the total RNA was extracted from RAW264.7 cultured under the respective conditions using a total RNAextraction reagent (ISOGEN, manufactured by Nippon Gene Co., Ltd.)according to the protocol attached to the reagent. The collected totalRNA was stored at −80° C.

b) When mouse bone marrow-derived primary cultured cells are cultured inthe presence of active vitamin D₃, a large number of TRAP-positivemultinucleated osteoclasts arise (Takahashi et al., Endocrinology,(1988) 122, 1373-1382).

A male ddY mouse at the age of 8 weeks was euthanized by cervicaldislocation under ether anesthesia and the femur and tibia wereresected. After soft tissues were removed, both ends of the femur ortibia were cut off. Then, α-MEM medium containing 10% fetal bovine serumwas injected into the bone marrow using a syringe barrel with a 25-gaugeinjection needle, and bone marrow cells were collected. After the numberof cells was counted, the cells were prepared at 5×10⁶ cells/ml in α-MEMmedium containing 10% fetal bovine serum. The resulting cell preparationwas plated in 60 wells of a 96-well plate at 100 μl/well, and activevitamin D₃ (manufactured by Sigma Co., Ltd.) was added thereto to give afinal concentration of 2×10⁻⁸ M, and the cells were cultured for 8 daysin a CO₂ incubator. Further, the culturing without the addition ofactive vitamin D₃ was performed in the same manner. Incidentally, themedium replacement and addition of active vitamin D₃ were performed ondays 3 and 6.

Thereafter, the total RNA was extracted from the cells cultured underthe respective conditions using a total RNA extraction reagent (ISOGEN,manufactured by Nippon Gene Co., Ltd.) according to the protocolattached to the reagent. The collected total RNA was stored at −80° C.until use.

Example 3 Acquisition of Sequence of Open Reading Frame (ORF) for MouseSiglec-15

a) Synthesis of First Strand cDNA

To 1 μg of the total RNA produced in a) of Example 2, 1 μl of 1 U/μlDNase I and 1 μl of 10× DNase I buffer (manufactured by Invitrogen,Inc.) were added, and then, the final volume was brought to 10 μl withH₂O. After a reaction was allowed to proceed at room temperature for 15minutes, 1 μl of 25 mM EDTA was added thereto and the resulting mixturewas heated at 65° C. for 10 minutes. From this solution, an 8 μl aliquotwas taken, and 1 μl of 50 μM oligo(dT)₂₀ primer and 1 μl of 10 mM dNTPswere added thereto, and the resulting mixture was heated at 65° C. for 5minutes and then incubated in ice. To this solution, 2 μl of 10×RTbuffer (manufactured by Invitrogen, Inc.), 4 μl of 25 mM MgCl₂, 2 μl of0.1 M dithiothreitol, 1 μl of RNase inhibitor (RNaseOUT, 40 U/μl,manufactured by Invitrogen, Inc.), and 1 μl of Superscript III reversetranscriptase (200 U/μl, manufactured by Invitrogen, Inc.) were addedand the total volume was brought to 20 μl. After a reaction was allowedto proceed at 50° C. for 50 minutes, the mixture was heated at 85° C.for 5 minutes and then incubated in ice for 1 minute. Thereafter, themixture was stored at −20° C.

b) PCR Reaction

Oligonucleotides having the sequences of: 5′-agaattccac cATGGAGGGGTCCCTCCAAC TC-3′ (mSiglec-15-EcoRI kozak-F: SEQ ID NO: 5 in the SequenceListing); and 5′-cgccgctcga gTTATTTCTC ATGGTGAATG AC-3′(mSiglec-15-XhoI-R: SEQ ID NO: 6 in the Sequence Listing) as primers foramplifying the ORF cDNA for mouse Siglec-15 by PCR were synthesizedaccording to a common procedure. The PCR was performed using thiscombination of primers and the cDNA produced in a) and high fidelitypolymerase (manufactured by Invitrogen, Inc.) according to a commonprocedure. The conditions for a thermal cycler were set as follows:after heating at 94° C. for 2 minutes, a temperature cycle of “94° C.for 0.5 minutes, 55° C. for 0.5 minutes and 68° C. for 1.5 minutes” wasrepeated 35 times, followed by heating at 68° C. for 5 minutes andincubating at 4° C. c) Cloning into pcDNA3.1(+) vector

The PCR reaction solution obtained in b) and pcDNA3.1(+) vector(manufactured by Invitrogen, Inc.) were treated with restriction enzymes(EcoRI, XhoI), followed by column purification, and then, a ligasereaction was performed according to a common procedure. Escherichia coliDH5α-T1 was transformed with the resulting vector and plated on a platecontaining ampicillin. From the thus obtained Escherichia coli colonies,transformed Escherichia coli containing the mouse Siglec-15/pcDNA3.1(+)plasmid was isolated.

The entire nucleotide sequence of the ORF cDNA inserted into theobtained plasmid was analyzed using a DNA sequencer, and as a result, itwas found to be the sequence represented by SEQ ID NO: 3 in the SequenceListing. This nucleotide sequence was the same as an ORF coding regionof a predicted sequence registered in NCBI GenBank database as “mouseCD33L3” (accession number: XM_(—)884636), and further, the amino acidsequence (SEQ ID NO: 4 in the Sequence Listing) encoded by thenucleotide sequence was 100% identical to the predicted amino acidsequence of the mouse CD33L3.

Example 4 Expression of mRNA for Siglec-15 Accompanying Mouse OsteoclastDifferentiation (Real-Time PCR Analysis)

a) To 1 μg of the total RNA produced in a) or b) of Example 2, 1 μl of 1U/μl DNase I and 1 μl of 10× DNase I buffer (manufactured by Invitrogen,Inc.) were added, and then, the final volume was brought to 10 μl withH₂O. After a reaction was allowed to proceed at room temperature for 15minutes, 1 μl of 25 mM EDTA was added thereto and the resulting mixturewas heated at 65° C. for 10 minutes. From this solution, an 8 μl aliquotwas taken, and 1 μl of 50 μM oligo(dT)₂₀ primer and 1 μl of 10 mM dNTPswere added thereto, and the resulting mixture was heated at 65° C. for 5minutes and then incubated in ice. To this solution, 2 μl of 10×RTbuffer (manufactured by Invitrogen, Inc.), 4 μl of 25 mM MgCl₂, 2 μl of0.1 M dithiothreitol, 1 μl of RNase inhibitor (RNaseOUT, 40 U/μl,manufactured by Invitrogen, Inc.), and 1 μl of Superscript III reversetranscriptase (200 U/μl, manufactured by Invitrogen, Inc.) were addedand the total volume was brought to 20 μl. After a reaction was allowedto proceed at 50° C. for 50 minutes, the mixture was heated at 85° C.for 5 minutes and then incubated in ice.

By using the thus produced single-stranded cDNA, real-time PCR wasperformed on a combination of the following primers and fluorescentlylabeled probes (TaqMan probe, manufactured by Applied Biosystems, Inc.).

Conditions for Real-Time PCR:

Primers for amplifying mouse Siglec-15: 5′-tcaggctcag gagtccaatt at-3′(TqM-mSiglec-15-F: SEQ ID NO: 7 in the Sequence Listing) and5′-ggtctagcct ggtactgtcc ttt-3′ (TqM-mSiglec-15-R: SEQ ID NO: 8 in theSequence Listing) TaqMan probe for detecting mouse Siglec-15:5′-Fam-atttgagcca gatgagtcct ccaggcca-TAMRA-3′(TqM-mSiglec-15-probe: SEQ ID NO: 9 in the Sequence Listing)Primers for amplifying mouse L32 ribosomal protein:5′-aagaagttca tcaggcacca gt-3′(TqM-mL32-F: SEQ ID NO: 10 in the Sequence Listing) and5′-cttgacattg tggaccagga ac-3′(TqM-mL32-R: SEQ ID NO: 11 in the Sequence Listing)TaqMan probe for detecting mouse L32 ribosomal protein:5′-Fam-aaacccagag gcattgacaa cagggtgc-TAMRA-3′(TqM-mL32-probe: SEQ ID NO: 12 in the Sequence Listing)

A real-time PCR analysis was performed using a real-time PCR system (ABIPrism 7700 Sequence Detector, manufactured by Perkin Elmer Japan AppliedBiosystems Division) under the following conditions. In the reaction,TaqMan Universal PCR Master Mix (manufactured by Applied Biosystems,Inc.) was used. First, distilled water was added to 25 pmol of eachprimer, 8 ng of single-stranded cDNA and 10 pmol of TaqMan probe tobring the final volume to 25 μl, and then, 25 μl of TaqMan Universal PCRMaster Mix was added thereto, whereby 50 μl of a reaction solution wasprepared. This reaction solution was heated at 50° C. for 2 minutes andthen heated at 95° C. for 10 minutes, and thereafter subjected to 40temperature cycles of “95° C. for 0.25 minutes and 60° C. for 1 minute”,whereby a real-time PCR analysis was performed. Incidentally, theexpression level of mRNA for mouse Siglec-15 was corrected by theexpression level of mRNA for L32 ribosomal protein.

As a result, the expression level of the Siglec-15 gene significantlyincreased in both cases where osteoclasts were induced by adding RANKLto RAW 264.7 and where osteoclasts were induced by adding active vitaminD₃ to mouse bone marrow-derived primary cultured cells (FIG. 3).

b) RAW 264.7 was prepared at 4.5×10⁴ cells/ml in α-MEM medium containing10% fetal bovine serum, and the resulting cell preparation was put in a75 cm² flask at 10 ml/flask, and then, human RANKL (manufactured byPeproTech Inc.) was added thereto to give a final concentration of 40ng/ml. The cells were cultured for 0, 1, 2, and 3 days in a CO₂incubator. Further, the culturing without the addition of human RANKLwas performed in the same manner.

After completion of the culturing, the total RNA was extracted from RAW264.7 cultured under the respective conditions using a total RNAextraction reagent (ISOGEN, manufactured by Nippon Gene Co., Ltd.)according to the protocol attached to the reagent. The collected totalRNA was stored at −80° C.

To 1 μg of the thus collected total RNA, 1 μl of 1 U/μl DNase I and 1 μlof 10× DNase I buffer (manufactured by Invitrogen, Inc.) were added, andthen, the final volume was brought to 10 μl with H₂O. After a reactionwas allowed to proceed at room temperature for 15 minutes, 1 μl of 25 mMEDTA was added thereto and the resulting mixture was heated at 65° C.for 10 minutes. From this solution, an 8 μl aliquot was taken, and 1 μlof 50 μM oligo(dT)₂₀ primer and 1 μl of 10 mM dNTPs were added thereto,and the resulting mixture was heated at 65° C. for 5 minutes and thenincubated in ice. To this solution, 2 μl of 10×RT buffer (manufacturedby Invitrogen, Inc.), 4 μl of 25 mM MgCl₂, 2 μl of 0.1 M dithiothreitol,1 μl of RNase inhibitor (RNaseOUT, 40 U/μl, manufactured by Invitrogen,Inc.), and 1 μl of Superscript III reverse transcriptase (200 U/μl,manufactured by Invitrogen, Inc.) were added and the total volume wasbrought to 20 μl. After a reaction was allowed to proceed at 50° C. for50 minutes, the mixture was heated at 85° C. for 5 minutes and thenincubated in ice.

By using the thus produced single-stranded cDNA, real-time PCR wasperformed on a combination of the following primers and fluorescentlylabeled probes (TaqMan probe, manufactured by Applied Biosystems, Inc.).

Conditions for Real-Time PCR:

Primers for amplifying mouse cathepsin K: 5′-ggcatctttc cagttttaca gc-3′(TqM-mcatK-F: SEQ ID NO: 13 in the Sequence Listing) and5′-gttgttctta ttccgagcca ag-3′(TqM-mcatK-R: SEQ ID NO: 14 in the Sequence Listing)TaqMan probe for detecting mouse cathepsin K:5′-Fam-atgtgaacca tgcagtgttg gtggtggg-TAMRA-3′(TqM-mcatK-probe: SEQ ID NO: 15 in the Sequence Listing)Primers for amplifying mouse TRAP: 5′-gaacttcccc agcccttact ac-3′(TqM-mTRAP-F: SEQ ID NO: 16 in the Sequence Listing) and5′-aactgctttt tgagccagga c-3′(TqM-mTRAP-R: SEQ ID NO: 17 in the Sequence Listing)TaqMan probe for detecting mouse TRAP:5′-Fam-ttgccagtca gcagcccaaa atgcct-TAMRA-3′(TqM-mTRAP-probe: SEQ ID NO: 18 in the Sequence Listing)Primers for amplifying mouse Siglec-15: 5′-tcaggctcag gagtccaatt at-3′(TqM-mSiglec-15-F: SEQ ID NO: 7 in the Sequence Listing) and5′-ggtctagcct ggtactgtcc ttt-3′ (TqM-mSiglec-15-R: SEQ ID NO: 8 in theSequence Listing) TaqMan probe for detecting mouse Siglec-15:5′-Fam-atttgagcca gatgagtcct ccaggcca-TAMRA-3′(TqM-mSiglec-15-probe: SEQ ID NO: 9 in the Sequence Listing)Primers for amplifying mouse L32 ribosomal protein:5′-aagaagttca tcaggcacca gt-3′(TqM-mL32-F: SEQ ID NO: 10 in the Sequence Listing) and5′-cttgacattg tggaccagga ac-3′(TqM-mL32-R: SEQ ID NO: 11 in the Sequence Listing)TaqMan probe for detecting mouse L32 ribosomal protein:5′-Fam-aaacccagag gcattgacaa cagggtgc-TAMRA-3′(TqM-mL32-probe: SEQ ID NO: 12 in the Sequence Listing)

A real-time PCR analysis was performed using a real-time PCR system (ABIPrism 7700 Sequence Detector, manufactured by Perkin Elmer Japan AppliedBiosystems Division) under the following conditions. In the reaction,TaqMan Universal PCR Master Mix (manufactured by Applied Biosystems,Inc.) was used. First, distilled water was added to 25 pmol of eachprimer, 8 ng of single-stranded cDNA and 10 pmol of TaqMan probe tobring the final volume to 25 μl, and then, 25 μl of TaqMan Universal PCRMaster Mix was added thereto, whereby 50 μl of a reaction solution wasprepared. This reaction solution was heated at 50° C. for 2 minutes andthen heated at 95° C. for 10 minutes, and thereafter subjected to 40temperature cycles of “95° C. for 0.25 minutes and 60° C. for 1 minute”,whereby a real-time PCR analysis was performed. Incidentally, theexpression level of mRNA for each gene was corrected by the expressionlevel of mRNA for L32 ribosomal protein.

As a result, the expression levels of cathepsin K and TRAP genes whichare known as marker molecules for osteoclasts significantly increasedfrom day 2 to day 3 after the addition of RANKL (FIGS. 4-A, B).Similarly, the expression level of the Siglec-15 gene also significantlyincreased from day 2 to day 3 after the addition of RANKL (FIG. 5). Fromthese results, it was revealed that the expression of the Siglec-15 geneincreases accompanying osteoclast differentiation, and particularly, theSiglec-15 gene is expressed strongly at a late differentiation stage.

Example 5 Production of Soluble Mouse Siglec-15 Protein ExpressionConstruct

A partial nucleic acid sequence encoding the extracellular domain ofmouse Siglec-15 protein is represented by SEQ ID NO: 19 in the SequenceListing and the amino acid sequence thereof is represented by SEQ ID NO:20 in the Sequence Listing. By utilizing such a partial sequence,soluble mouse Siglec-15 protein can be produced in a culture supernatantof an animal cell or the like.

a) Amplification of Soluble Mouse Siglec-15 Gene by PCR

Oligonucleotides having the sequences of: 5′-ggggacaagt ttgtacaaaaaagcaggctt caccATGGAG GGGTCCCTCC AACTC-3′ (mSiglec-15-ECD-F: SEQ ID NO:21 in the Sequence Listing); and 5′-ggggaccact ttgtacaaga aagctgggtcTCCGGGGGCG CCGTGGAAGC GGAAC-3′ (mSiglec-15-ECD-R: SEQ ID NO: 22 in theSequence Listing) as primers for amplifying the mouse Siglec-15extracellular domain cDNA by PCR were synthesized according to a commonprocedure. Incidentally, these primers were designed, as amplificationprimers for producing a gateway entry clone, such that an attB1 sequenceis added to mSiglec-15-ECD-F and an attB2 sequence is added tomSiglec-15-ECD-R. The PCR was performed using this combination ofprimers and the mouse Siglec-15/pcDNA3.1(+) plasmid produced in Example3 as a template according to a common procedure. The conditions for athermal cycler were set as follows: after heating at 94° C. for 2minutes, a temperature cycle of “94° C. for 0.5 minutes, 55° C. for 0.5minutes and 68° C. for 1.5 minutes” was repeated 15 times, followed byheating at 68° C. for 5 minutes and incubating at 4° C.

b) Production of Entry Clone by Gateway BP Reaction

An entry clone into which the mouse Siglec-15 extracellular domain cDNAwas integrated by the Gateway technology (Invitrogen, Inc.) employing alambda phage site-specific recombination system was produced by thefollowing method. First, a BP reaction using BP Clonase was performedbetween the PCR product having an attB sequence at both ends produced ina) and pDNOR221 (manufactured by Invitrogen, Inc.) which is a donorvector having an attP sequence. By using this reaction solution,Escherichia coli DH10B was transformed, and colony PCR was performed fordrug-resistant clones, and the size of inserts was confirmed. Then, fora clone confirmed to have an insert with a correct size, a sequenceanalysis of the total DNA sequence of the insert was performed. As aresult, an entry clone which is completely identical to the targetnucleic acid sequence (SEQ ID NO: 19 in the Sequence Listing) encodingthe extracellular domain of mouse Siglec-15 protein was obtained.

c) Production of Expression Clone by Gateway LR Reaction

An expression clone into which the mouse Siglec-15 extracellular domaincDNA was integrated by the Gateway technology (Invitrogen, Inc.)employing a lambda phage site-specific recombination system was producedby the following method. The entry clone produced in b) contains aninsert having an attL sequence at both ends. An LR reaction using LRClonase was performed between this entry clone and two types ofdestination vectors having an attR sequence. Incidentally, as thedestination vectors, two types of destination vectors: pDONM designedsuch that a V5 epitope tag and a 6×His tag are added to the C terminusof the insert; and phIgFc designed such that a human Fc tag is added tothe C terminus of the insert were used. By using the reaction solutionobtained by the LR reaction, Escherichia coli DH10B was transformed, andcolony PCR was performed for the obtained drug-resistant clones, and thesize of inserts was confirmed. Then, for a clone confirmed to have aninsert with a correct size, a sequence analysis of both ends from theinsert side to the vector side was performed.

Primer Sequences for Sequence Analysis

5′-tgcgtgaagg tgcagggcag-3′(mSiglec-15-ECD-seq-upstm: SEQ ID NO: 23 in the Sequence Listing) and5′-cctcgcctgg tcgggtc-3′ (mSiglec-15-ECD-seq-dnstm: SEQ ID NO: 24 in theSequence Listing)

As a result of the sequence analysis, expression clones (soluble mouseSiglec-15/pDONM and soluble mouse Siglec-15/phIgFc) in which correctrecombination occurred were obtained for both pDONM and phIgFc,respectively. By transfecting the soluble mouse Siglec-15/pDONM into ananimal cell or the like, mRNA having the base sequence represented bySEQ ID NO: 25 in the Sequence Listing is transcribed and translated intoa protein (mouse Siglec-15-His) having the amino acid sequencerepresented by SEQ ID NO: 26 in the Sequence Listing. Further, bytransfecting the soluble mouse Siglec-15/phIgFc into an animal cell orthe like, mRNA having the base sequence represented by SEQ ID NO: 27 inthe Sequence Listing is transcribed and translated into a protein (mouseSiglec-15-Fc) having the amino acid sequence represented by SEQ ID NO:28 in the Sequence Listing.

Example 6 Examination of Optimal Culture Time for Producing SolubleMouse Siglec-15 Protein a) Expression of Protein Using 293-F Cells

The two types of expression plasmids (soluble mouse Siglec-15/pDONM andsoluble mouse Siglec-15/phIgFc) obtained in Example 5 were prepared inan amount of about 100 μg, respectively. 50 μg of each of the preparedplasmids was mixed with Opti-MEM (manufactured by Invitrogen, Inc.),followed by filter sterilization. Then, 64 μl of a transfection reagent293fectin (manufactured by Invitrogen, Inc.) was added thereto, and theresulting mixture was incubated at room temperature for 25 minutes. Eachof the thus obtained mixtures was added to FreeStyle 293-F cells(manufactured by Invitrogen, Inc.) cultured in a shake flask such thatthe cell density reached 1.0×10⁶ cells/ml×50 ml in FreeStyle 293Expression Medium (manufactured by Invitrogen, Inc.), and the cells weresubjected to rotary culture (125 rotations/min) at a CO₂ concentrationof 8.0% for 96 hours (4 days) at 37° C. A small portion of the culturesolution was collected at 24-hour intervals (culture time: 0, 24, 48,72, 96 hours), and centrifuged to prepare a culture supernatant. It isconsidered that in the thus prepared culture supernatants, a protein inwhich a V5 epitope tag and a 6×His tag have been added to the C-terminalside of the mouse Siglec-15 extracellular domain (mouse Siglec-15-His)and a protein in which a human Fc tag has been added to the C-terminalside of the mouse Siglec-15 extracellular domain (mouse Siglec-15-Fc)are expressed, respectively.

b) Change in Expression Level with Culture Time of MouseSiglec-15-his-Expressing 293F Cells

By using the culture solution (culture time: 0, 24, 48, 72, 96 hours)samples of mouse Siglec-15-His-expressing 293F cells prepared in a) anda commercially available His tag-containing protein, recombinant humanosteoprotegerin/his (OPG-Fc-His) (manufactured by R&D systems, Inc.),the expression levels were analyzed by SDS-polyacrylamideelectrophoresis under reducing conditions and Western blotting. That is,to 5 μl of a sample obtained by concentrating each culture solution by20-fold using Microcon YM-10 (manufactured by Millipore Co., Ltd.) or 5μl of an OPG-Fc-His solution, an equivalent amount of an SDS-treatmentsolution (10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA, 2.5% SDS,0.1% bromophenol blue, and 5% 2-mercaptoethanol) was added, and theresulting mixture was heated at 95° C. for 10 minutes. 0.8 μl of each ofthe thermally treated samples was used for SDS-polyacrylamideelectrophoresis. As a gel for electrophoresis, an 8-25% polyacrylamidegradient gel (manufactured by Amersham Biosciences, Inc.) was used, andthe electrophoresis was performed using PhastSystem (manufactured byAmersham Biosciences, Inc.). Further, as molecular weight markers, ECLDualVue Western Blotting Markers (manufactured by Amersham Biosciences,Inc.) were used. After completion of the electrophoresis, the protein inthe gel was transferred (blotted) to a PVDF membrane (Hybond-P,manufactured by Amersham Biosciences, Inc.) using PhastTransfer Semi-dryTransfer Kit (manufactured by Amersham Biosciences, Inc.) andPhastSystem. This PVDF membrane was transferred in 10 ml of a blockingagent (BlockAce, manufactured by Snow Brand Milk Products, Co., Ltd.)containing 0.1% Tween 20 and gently shaken at room temperature for 1hour. To this blocking solution, 5 μl of an S-protein HRP solution (ECLDualVue Western Blotting Markers, manufactured by Amersham Biosciences,Inc.) and 2 μl of an anti-6-His-HRP antibody (PentaHis HRP Conjugatekit, manufactured by Qiagen, Inc.) were added and the membrane in thesolution was gently shaken at room temperature for an additional 1 hour.This PVDF membrane was washed 4 times by gently shaking it in 50 mL ofphosphate-buffered saline (PBS) containing 0.01% Tween 20 for 5 minutes.After washing, the PVDF membrane was treated according to the protocolattached to an ECL detection kit (ECL Western blotting detectionreagents and analysis system, manufactured by Amersham Biosciences,Inc.) to develop the color of the band of the His tag-containingprotein, and the developed color was detected using an ECL Mini-Camera(manufactured by Amersham Biosciences, Inc.) and Polaroid film (Polapan3200B, manufactured by Polaroid, Inc.). The results are shown in FIG. 6.From these results, a 96-hour culture time was selected as the culturetime for 293F cells which produced the highest concentration of aprotein (mouse Siglec-15-His) which has a molecular weight of about 35kDa and reacts with an anti-6-His-HRP antibody.

c) Change in Expression Level with Culture Time of MouseSiglec-15-Fc-Expressing 293F Cells

By using the culture solution (culture time: 0, 24, 48, 72, 96 hours)samples of mouse Siglec-15-Fc-expressing 293F cells prepared in a) andhuman IgG (manufactured by Sigma Co., Ltd), the expression levels wereanalyzed by SDS-polyacrylamide electrophoresis under non-reducingconditions and Western blotting. That is, to 5 μl of each culturesolution sample or 5 μl of a human IgG solution, an equivalent amount ofan SDS-treatment solution was added, and the resulting mixture washeated at 95° C. for 10 minutes. In the same manner as the methoddescribed in the above b) using 0.8 μl of each of the thermally treatedsamples, SDS-polyacrylamide electrophoresis, transfer (blotting) to aPVDF membrane, and blocking of the PVDF membrane were performed. To thePVDF membrane after blocking, 5 μl of an S-protein HRP solution (ECLDualVue Western Blotting Markers, manufactured by Amersham Biosciences,Inc.) and 2 μl of an anti-human IgG-Fc-HRP antibody (Anti-Humam IgG (Fcspecific) Peroxidase Conjugate, manufactured by Sigma Co., Ltd) wereadded and the membrane in the solution was gently shaken at roomtemperature for an additional 1 hour. After washing was performed in thesame manner as the method described in the above b), the developed colorof the band of the Fc-containing protein was detected. The results areshown in FIG. 7. From these results, a 96-hour culture time was selectedas the culture time for 293F cells which produced the highestconcentration of a protein (mouse Siglec-15-Fc) which has a molecularweight of about 110 kDa and reacts with an anti-human Fc antibody.

Example 7 Large-Scale Preparation of Culture Solution Containing SolubleMouse Siglec-15 Protein Using 293-F Cells

The two types of expression plasmids (soluble mouse Siglec-15/pDONM andsoluble mouse Siglec-15/phIgFc) obtained in Example 5 were prepared inan amount of about 5 mg, respectively. Incidentally, in the purificationof plasmids from Escherichia coli cultured on a large scale, InvitrogenPureLink HiPure Plasmid Gigaprep Kit (manufactured by Invitrogen, Inc.)was used. The thus prepared plasmids were mixed with Opti-MEM(manufactured by Invitrogen, Inc.), followed by filter sterilization.Then, 10 ml of a transfection reagent 293fectin (manufactured byInvitrogen, Inc.) was added thereto, and the resulting mixture wasincubated at room temperature for 20 minutes. Each of the thus obtainedmixtures was added to FreeStyle 293-F cells (manufactured by Invitrogen,Inc.) cultured in Erlenmeyer flasks such that the cell density reached1.1×10⁶ cells/ml×5 L (1 L/flask×5 flasks) in FreeStyle 293 ExpressionMedium (manufactured by Invitrogen, Inc.). After the cells weresubjected to rotary culture (125 rotations/min) at a CO₂ concentrationof 8.0% for 96 hours (4 days) at 37° C., the culture solution wascollected and centrifuged to prepare a culture supernatant. It isconsidered that in the thus prepared culture supernatants, a protein inwhich a V5 epitope tag and a 6×His tag have been added to the C-terminalside of the mouse Siglec-15 extracellular domain (mouse Siglec-15-His)and a protein in which a human Fc tag has been added to the C-terminalside of the mouse Siglec-15 extracellular domain (mouse Siglec-15-Fc)are expressed, respectively.

Example 8 Purification of Mouse Siglec-15-his a) HisTrap HP ColumnChromatography

To 2 L of the culture solution of mouse Siglec-15-His-expressing 293Fcells prepared in Example 7, 225 mL of 10×buffer (500 mM Tris, 1.5 MNaCl, 200 mM imidazole, pH 8.0) was added, and the resulting mixture wasstirred well and filtered through a Sterivex-GV filter (manufactured byMillipore Co., Ltd.). This culture solution was applied to a columnwhich comprised three HisTrap HP 5 ml columns (manufactured by AmershamBiosciences, Inc.) connected in series and was previously treated with apyrogen removing agent PyroCLEAN (manufactured by ALerCHEK, Inc.) andwashed with distilled water for injection at a flow rate of 2 ml/min.After the column was washed with 60 ml of 50 mM Tris-HCl buffer (pH 8.0)containing 300 mM NaCl at a flow rate of 1 ml/min, a protein adsorbedonto the column was eluted with 50 ml of 50 mM Tris-HCl buffer (pH 8.0)containing 300 mM NaCl and 500 mM imidazole at a flow rate of 1 ml/min.The eluate was fractionated at 1 ml per fraction into mini-sorp tubes(manufactured by Nunc, Inc.) to which 10 μl of 10% Tween 20 hadpreviously been added. After about 20 ml of a solution obtained bycombining the fractions (fractions 14 to 20) containing the elutedprotein was concentrated to 2.5 ml with a centrifugal membraneconcentrator Amicon Ultra-15 (manufactured by Millipore Co., Ltd.), theconcentrate was applied to a PD-10 desalting column (manufactured byAmersham Biosciences, Inc.) which was previously equilibrated withphosphate-buffered saline containing 0.01% Tween 20 (T-PBS), followed byelution with T-PBS, whereby 3.5 ml of a sample whose solvent wasreplaced with T-PBS was obtained.

b) Resource Q Column Chromatography

To 3.5 ml of the sample which was purified by HisTrap HP columnchromatography and whose solvent was replaced with TBS-P, 22.5 ml of 50mM Tris-HCl buffer (pH 7.5) containing 0.1% CHAPS was added and theresulting mixture was stirred. Then, the mixture was centrifuged at 4°C. for 30 minutes at 3,000 rpm and the precipitate was removed. Afterthe resulting supernatant was filtered through a Millex-GV filter(manufactured by Millipore Co., Ltd.), the filtrate was applied to aResource Q 6 ml column (manufactured by Amersham Biosciences, Inc.)which was previously equilibrated with 50 mM Tris-HCl buffer (pH 7.5)containing 0.1% CHAPS at a flow rate of 1 ml/min. Thereafter, the columnwas washed with this buffer at a flow rate of 1 ml/min and a proteinfraction which was not adsorbed onto the column was collected. A proteinadsorbed onto the column was eluted with 50 mM Tris-HCl buffer (pH 7.5)containing 0.1% CHAPS and 1 M NaCl at a flow rate of 1 ml/min. After26.5 ml of the fraction which was not adsorbed onto the column wasconcentrated to 2.0 ml with a centrifugal membrane concentrator AmiconUltra-15 (manufactured by Millipore Co., Ltd.), the concentrate wascentrifuged at 4° C. for 10 minutes at 3,000 rpm and the precipitate wasremoved. The supernatant after centrifugation was cryopreserved at −80°C. until use. The above-mentioned purification procedure (HisTrap HPcolumn chromatography and Resource Q column chromatography) wasperformed twice by repeating it.

c) Detection and Purity Assay of Purified Mouse Siglec-15-his

By using a sample prepared by the above-mentioned purification procedure(HisTrap HP column chromatography and Resource Q column chromatography),SDS-polyacrylamide electrophoresis under reducing conditions and silverstaining were performed. That is, to 5 μl of each of the samplespurified by the respective purification steps, an equivalent amount ofan SDS-treatment solution was added, and the resulting mixture wasthermally treated at 95° C. for 10 minutes. 0.3 μl of each of thethermally treated samples was used for SDS-polyacrylamideelectrophoresis. The electrophoresis procedure was performed in the samemanner as in the above-mentioned b) of Example 6 except that RainbowMolecular Weight Markers (manufactured by Amersham Biosciences, Inc.)were used as the molecular weight markers. After completion of theelectrophoresis, silver staining was performed using PhastGel Silver Kit(manufactured by Amersham Biosciences, Inc.) and PhastSystem. Theresults are shown in FIG. 8. It was shown that a protein having amolecular weight of about 35 kDa (mouse Siglec-15-His) was efficientlypurified and concentrated in the protein fraction which was not adsorbedonto the Resource Q column.

Electrophoresis was performed under the same conditions except that ECLDualVue Western Blotting Markers (manufactured by Amersham Biosciences,Inc.) were used as the molecular weight markers, and the protein in thegel was transferred (blotted) to a PVDF membrane (Hybond-P, manufacturedby Amersham Biosciences, Inc.) using PhastTransfer Semi-dry Transfer Kit(manufactured by Amersham Biosciences, Inc.) and PhastSystem. This PVDFmembrane was gently shaken in 10 ml of a blocking agent (BlockAce,manufactured by Snow Brand Milk Products, Co., Ltd.) containing 0.1%Tween 20 at room temperature for 1 hour. To this blocking solution, 10μl of S-protein HRP (manufactured by Amersham Biosciences, Inc.) and 10μl of an anti-V5-HRP antibody (Monoclonal Antibody to Pk-TAG-HRP,manufactured by Acris Antibodies GmbH) were added and the membrane inthe solution was gently shaken at room temperature for an additional 1hour. The PVDF membrane was washed 4 times by gently shaking it in 50 mLof phosphate-buffered saline (PBS) containing 0.01% Tween 20 for 5minutes. After washing, the PVDF membrane was treated according to theprotocol attached to an ECL detection kit (manufactured by AmershamBiosciences, Inc.) to develop the color of the band of the protein, andthe developed color was detected using an ECL Mini-Camera (manufacturedby Amersham Biosciences, Inc.) and Polaroid film (Polapan 3200B,manufactured by Polaroid, Inc.). The results are shown in FIG. 9. Alsofrom these results, it could be confirmed that a protein which has amolecular weight of about 35 kDa (mouse Siglec-15-His) and reacts withan anti-V5-HRP antibody was efficiently purified and concentrated in theprotein fraction which was not adsorbed onto the Resource Q column.

d) Measurement of Protein Concentration of Purified Mouse Siglec-15-his

For the purified mouse Siglec-15-His (the protein fraction which was notadsorbed onto the Resource Q column), the protein concentration wasmeasured with a DC-Protein Assay kit (manufactured by Bio-RadLaboratories, Inc.) using bovine serum albumin as a standard sample. Asshown in Table 1, a total of 1.66 mg of purified mouse Siglec-15-Hisprotein was obtained by performing the purification procedure twice.

TABLE 1 Protein Conc. (mg/ml) Sample Vol. (ml) Total protein (mg) 1st0.475 2.0 0.95 2nd 0.354 2.0 0.71 Total 1.66

Example 9 Purification of Mouse Siglec-15-Fc a) HiTrap Protein a ColumnChromatography

1.8 L of the culture solution of mouse Siglec-15-Fc-expressing 293Fcells prepared in Example 7 was filtered through a Sterivex-GV filter(manufactured by Millipore Co., Ltd.), and then, the filtrate wasapplied to a HiTrap Protein A 5 ml column (manufactured by AmershamBiosciences, Inc.) which had previously been equilibrated withDulbecco's PBS (D-PBS, manufactured by Invitrogen, Inc.) at a flow rateof 5 ml/min. After the column was washed with D-PBS at a flow rate of 5ml/min, a protein adsorbed onto the column was eluted with 50 ml of 0.1M sodium citrate buffer (pH 3.0) at a flow rate of 5 ml/min. The eluatewas fractionated at 5 ml per fraction into mini-sorp tubes (manufacturedby Nunc, Inc.), and immediately thereafter, 1.3 ml of 1 M Tris was addedthereto to neutralize the eluate. After a solution obtained by combiningthe fractions (fractions 1 and 2) in which the eluted protein wasdetected was concentrated to 2.5 ml with a centrifugal membraneconcentrator Amicon Ultra-15 (manufactured by Millipore Co., Ltd.), theconcentrate was applied to a PD-10 desalting column (manufactured byAmersham Biosciences, Inc.) which was previously equilibrated withOtsuka Physiological Saline for Injection (TO-SS, manufactured by OtsukaPharmaceutical Co., Ltd.) containing 0.01% Tween 20, followed by elutionwith TO-SS, whereby 3.5 ml of a sample whose solvent was replaced withTO-SS was obtained. This sample was cryopreserved at −80° C. until use.By using 2.9 L of a culture solution of 293F cells, the samepurification procedure was performed once again by repeating it.

b) Detection and Purity Assay of Purified Mouse Siglec-15-Fc

By using a sample prepared by the above-mentioned purificationprocedure, SDS-polyacrylamide electrophoresis under reducing conditionsand silver staining were performed. That is, to 5 μl of each of thesamples purified by the respective purification steps, an equivalentamount of an SDS-treatment solution was added, and the resulting mixturewas heated at 95° C. for 10 minutes. 0.3 μl of a sample obtained bydiluting each of the thermally treated samples to 1/300 or 1/900 with ahalf concentration of the SDS-treatment solution was used forSDS-polyacrylamide electrophoresis. The electrophoresis and silverstaining were performed in the same manner as the purity assay of mouseSiglec-15-His described in c) of Example 8. The results are shown inFIG. 10 along with the results of examining preliminary purificationconditions on a small scale (the pH of the applied culture solution was8.9 or 7.0). It was shown that a protein having a molecular weight ofabout 55 kDa (mouse Siglec-15-Fc) was efficiently purified andconcentrated in the protein fraction which was eluted from the HiTrapProtein A column.

c) Measurement of Protein Concentration of Purified Mouse Siglec-15-Fc

For the purified mouse Siglec-15-Fc (the protein fraction eluted fromthe PD-10 desalting column), the protein concentration was measured witha DC-Protein Assay kit (manufactured by Bio-Rad Laboratories, Inc.)using bovine serum albumin as a standard sample. As shown in Table 2, atotal of 92 mg of purified mouse Siglec-15-Fc protein was obtained byperforming the purification procedure twice.

TABLE 2 Protein Conc. (mg/ml) Sample Vol. (ml) Total protein (mg) 1st8.0 3.5 28 2nd 18.5 3.5 64 Total 92

Example 10 Production of Rabbit Anti-Mouse Siglec-15 Polyclonal Antibody(Immunization of Rabbit) a) Preparation of Antigen

The mouse Siglec-15-Fc protein produced in Example 9 was prepared at 100μg/0.5 ml, and an equivalent amount of an adjuvant was added thereto andan emulsion was produced using a glass syringe. As the adjuvant,Freund's complete adjuvant (FCA, Manufactured by Difco Laboratories,Inc.) was used only for the first immunization, and Freund's incompleteadjuvant (FICA, Manufactured by Difco Laboratories, Inc.) was used forthe second and subsequent immunizations.

b) Immunization of Rabbit

Three rabbits (Japanese white female rabbits with a body weight of 3 kg)were used as immunized animals. Incidentally, the blood was collectedbefore immunization, and 1 ml of pre-immune serum was obtained perrabbit. The emulsion obtained in a) was injected subcutaneously andintradermally using a 27 G injection needle at 1 ml/rabbit. Immunizationwas performed a total of 8 times every 14 days after the firstimmunization. The whole blood was collected after 7 days from the dateof 8th immunization, and 76 to 79 ml of antiserum was obtained perrabbit. The antibody titers in the pre-immune serum and the antiserumwere confirmed by an ELISA method using an immobilized antigen. As aresult, an increase in antibody titer in the antiserum was confirmed inall the three rabbits. The antiserum was stored at −20° C. until use.

Example 11 Purification of Anti-Mouse Siglec-15 Polyclonal Antibody a)HiTrap Protein A Column Chromatography

To 20 ml of each of the three rabbit antiserum lots prepared in Example10, 20 ml of Dulbecco's PBS (D-PBS, manufactured by Invitrogen, Inc.)was added and mixed, and the resulting mixture was filtered through aSterivex-GV filter (manufactured by Millipore Co., Ltd.). Then, thefiltrate was applied to a HisTrap Protein A 5 ml column (manufactured byAmersham Biosciences, Inc.) which had previously been equilibrated withD-PBS at a flow rate of 2 ml/min. After the column was washed with 37.5ml of D-PBS at a flow rate of 2.5 ml/min, a protein adsorbed onto thecolumn was eluted with 50 ml of 0.1 M sodium citrate buffer (pH 3.0) ata flow rate of 2.5 ml/min. The eluate was fractionated at 2.5 ml perfraction into mini-sorp tubes (manufactured by Nunc, Inc.), andimmediately thereafter, 0.65 ml of 1 M Tris was added thereto toneutralize the eluate. After about 10 ml of a solution obtained bycombining the fractions (fractions 2 to 5) containing the eluted proteinwas concentrated to 2.5 ml with a centrifugal membrane concentratorAmicon Ultra-15 (manufactured by Millipore Co., Ltd.), the concentratewas applied to a PD-10 desalting column (manufactured by AmershamBiosciences, Inc.) which had previously been equilibrated with OtsukaPhysiological Saline for Injection (TO-SS) containing 0.01% Tween 20,followed by elution with TO-SS, whereby 3.5 ml of a sample whose solventwas replaced with TO-SS was obtained. The thus prepared sample wascryopreserved at −80° C. until use.

b) Detection and Purity Assay of Anti-Mouse Siglec-15 PolyclonalAntibody

By using the samples (three lots, Nos. 1, 2, and 3) prepared by thepurification procedure described in the above a), SDS-polyacrylamideelectrophoresis under reducing conditions and silver staining wereperformed. That is, to 5 μl of each of the samples purified by therespective purification steps, an equivalent amount of an SDS-treatmentsolution (10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA, 2.5% SDS,0.1% bromophenol blue, and 5% 2-mercaptoethanol) was added, and theresulting mixture was heated at 95° C. for 10 minutes. 0.3 μl of asample obtained by diluting each of the thermally treated samples to1/100, 1/300 or 1/900 with a half concentration of the SDS-treatmentsolution was used for SDS-polyacrylamide electrophoresis. Theelectrophoresis and silver staining were performed in the same manner asthe purity assay of mouse Siglec-15-His described in c) of Example 8. Itwas shown that an IgG protein composed of a heavy chain having amolecular weight of about 45 kDa and a light chain having a molecularweight of about 21 kDa was efficiently purified and concentrated in theprotein fraction which was eluted from the PD-10 desalting column.

c) Measurement of Protein Concentration of Purified Anti-Mouse Siglec-15Polyclonal Antibody

For the purified anti-mouse Siglec-15 polyclonal antibody (the proteinfraction eluted from the PD-10 desalting column), the proteinconcentration was measured with a DC-Protein Assay kit (manufactured byBio-Rad Laboratories, Inc.) using bovine IgG as a standard sample. Asshown in Table 3, 100 to 170 mg of the anti-mouse Siglec-15 polyclonalantibody could be purified in each of the lots, Nos. 1 to 3.

TABLE 3 Protein Conc. (mg/ml) Sample Vol. (ml) Total protein (mg) No. 139.9 3.5 140 No. 2 28.8 3.5 100 No. 3 48.7 3.5 170

d) Examination of Reactivity of Purified Anti-Mouse Siglec-15 PolyclonalAntibody to Siglec-15 Extracellular Domain

A test for confirming that the anti-mouse Siglec-15 polyclonal antibodyprepared in the above item a) binds not only to an Fc tag but also tothe extracellular domain of Siglec-15 protein was performed. To 5 μl ofthe purified mouse Siglec-15-His sample (Example 8) or 5 μl of thepurified mouse Siglec-15-Fc sample, an equivalent amount of anSDS-treatment solution (with or without the addition of 5%2-mercaptoethanol) was added, and the resulting mixture was heated at95° C. for 10 minutes. 0.3 μl of each of the thermally treated sampleswas used for SDS-polyacrylamide electrophoresis, and electrophoresis andtransfer (blotting) to a PVDF membrane were performed in the same manneras the method described in the above b) of Example 6. This PVDF membranewas gently shaken in 8 ml of a blocking agent (BlockAce, manufactured bySnow Brand Milk Products, Co., Ltd.) containing 0.1% Tween 20 at roomtemperature for 1 hour. To this blocking solution, 1.6 μl of theanti-mouse Siglec-15 polyclonal antibody No. 1 (Table 3) was added, andthe PVDF membrane in the solution was gently shaken at room temperaturefor an additional 1 hour. This PVDF membrane was washed 4 times bygently shaking it in 50 mL of PBS containing 0.01% Tween 20 for 5minutes. The washed PVDF membrane was immersed in 8 ml of AntibodyDiluent ECL Advance Blocking Agent (ECL Advance Western BlottingDetection Kit, manufactured by Amersham Biosciences, Inc.), andanti-rabbit IgG-HRP (manufactured by Amersham Biosciences, Inc.) wasadded thereto to give a final concentration of 1/200,000. Then, 0.8 μlof an S-protein HRP solution (ECL DualVue Western Blotting Markers,manufactured by Amersham Biosciences, Inc.) was added thereto, and themembrane in the solution was gently shaken at room temperature for anadditional 1 hour. This PVDF membrane was washed 4 times by gentlyshaking it in 50 mL of PBS containing 0.01% Tween 20 for 5 minutes.After washing, the PVDF membrane was treated according to the protocolattached to ECL Advance Western Blotting Detection Kit (manufactured byAmersham Biosciences, Inc.), and the developed color of the band of theprotein was detected using an ECL Mini-Camera (manufactured by AmershamBiosciences, Inc.) and Polaroid film (Polapan 3200B, manufactured byPolaroid, Inc.). The results are shown in FIG. 11. From these results,it was shown that the purified anti-mouse Siglec-15 polyclonal antibodyalso binds to mouse Siglec-15-His, and it could be confirmed that theanti-mouse Siglec-15 polyclonal antibody binds not only to an Fc tag butalso to the extracellular domain of the Siglec-15 protein. The same testwas performed by repeating it, and it was confirmed that the anti-mouseSiglec-15 polyclonal antibodies No. 2 and No. 3 also bind to mouseSiglec-15-His.

Example 12 Purification of Pre-Immune Rabbit IgG

Blood had previously been collected from each of the three rabbits usedin Example 10, before initiation of immunization with mouseSiglec-15-Fc, and pre-immune serum was prepared therefrom. After a 0.8ml aliquot of each of these serum samples was mixed with one another,2.4 ml of Dulbecco's PBS (D-PBS, manufactured by Invitrogen, Inc.) wasadded thereto, and the resulting mixture was filtered through aMillex-GV filter (manufactured by Millipore Co., Ltd.). Then, theresulting serum sample was applied to a HiTrap Protein A 5 ml column(manufactured by Amersham Biosciences, Inc.) which had previously beenequilibrated with D-PBS at a flow rate of 1 ml/min. After the column waswashed with 50 ml of D-PBS at a flow rate of 2.5 ml/min, a proteinadsorbed onto the column was eluted with 50 ml of 0.1 M sodium citratebuffer (pH 3.0) at a flow rate of 2.5 ml/min. The eluate wasfractionated at 2.5 ml per fraction into mini-sorp tubes (manufacturedby Nunc, Inc.), and immediately thereafter, 0.65 ml of 1 M Tris wasadded thereto to neutralize the eluate. After a solution obtained bycombining the fractions (fractions 2 to 4) containing the eluted proteinwas concentrated to 2.5 ml with a centrifugal membrane concentratorAmicon Ultra-15 (manufactured by Millipore Co., Ltd.), the concentratewas applied to a PD-10 desalting column (manufactured by AmershamBiosciences, Inc.) which was previously equilibrated with OtsukaPhysiological Saline for Injection (TO-SS) containing 0.01% Tween 20,followed by elution with TO-SS, whereby 3.5 ml of a sample whose solventwas replaced with TO-SS was obtained. The thus purified pre-immunerabbit IgG sample was subjected to polyacrylamide electrophoresis andsilver staining by the method described in the above c) of Example 8 toconfirm that the IgG protein was sufficiently purified, and then theprotein concentration was measured. The thus purified sample wascryopreserved at −80° C. until use.

Example 13 Preparation of Affinity Column Having Mouse Siglec-15-FcImmobilized Thereon

After 0.54 ml of the solvent of the 18.5 mg/ml purified mouseSiglec-15-Fc solution described in Example 9 (a total of 10 mg ofprotein) was replaced with a coupling buffer (0.2 M NaHCO₃, 0.5 M NaCl,pH 8.3) using a PD-10 desalting column, the resulting solution wasconcentrated to 1 ml using a centrifugal membrane concentrator AmiconUltra-4 (manufactured by Millipore Co., Ltd.). After isopropanol in anNHS-activated HiTrap column (1 ml, manufactured by Amersham Biosciences,Inc.) was replaced with 1 mM hydrochloric acid, 1 ml of a couplingbuffer containing 10 mg/ml mouse Siglec-15-Fc was injected into thecolumn using a syringe. After a reaction was allowed to proceed at roomtemperature for 30 minutes, in order to inactivate excess active groups,6 ml of a blocking buffer (an ethanolamine buffer containing 0.5 M NaCl,pH 8.3), 6 ml of a washing buffer (a sodium acetate buffer containing0.5 M NaCl, pH 4.0), and 6 ml of the blocking buffer were injected insequence according to the protocol of Amersham Biosciences, Inc., andthen, the column was left at room temperature for 30 minutes.Thereafter, 6 ml of the washing buffer, 6 ml of the blocking buffer, and6 ml of the washing buffer were injected into the column in sequenceagain, and finally, the buffer in the column was replaced with 50 mMTris-HCl buffer (pH 7.0) containing 1 M NaCl and 0.01% Tween 20. Thiscolumn was stored at 4° C. until use.

Example 14 Purification of Anti-Mouse Siglec-15 Polyclonal Antibody withAffinity Column a) Affinity Column Chromatography

To 2 ml of each of the purified anti-mouse Siglec-15 polyclonalantibodies Nos. 1, 2 and 3 prepared in Example 11, 8 ml of an ApplyBuffer (10 mM Tris-HCl buffer containing 0.15 M NaCl, pH 7.2) was added,and the resulting mixture was applied to the affinity column (Example13) which had previously been equilibrated with the Apply Buffer at aflow rate of 0.25 ml/min. After the column was washed with 5 ml of theApply Buffer at a flow rate of 0.25 ml/min, first, a protein adsorbedonto the column was eluted with 5 ml of 0.1 M glycine hydrochloridebuffer (pH 2.7) containing 0.5 M NaCl at a flow rate of 0.25 ml/min, andsubsequently, a protein adsorbed onto the column was eluted with 5 ml of0.1 M sodium citrate buffer (pH 2.0) containing 0.5 M NaCl at a flowrate of 0.25 ml/min. The chromatogram of the anti-mouse Siglec-15polyclonal antibody No. 3 purified with the affinity column is shown inFIG. 12. The eluate was fractionated at 0.5 ml per fraction intomini-sorp tubes (manufactured by Nunc, Inc.), and immediatelythereafter, 16 μl of 1 M Tris was added to 0.5 ml of each fractioneluted with the glycine hydrochloride buffer, and 150 μl of 1 M Tris wasadded to 0.5 ml of each fraction eluted with the sodium citrate bufferto neutralize the eluate. Most of the anti-mouse Siglec-15 polyclonalantibody was eluted with the 0.1 M glycine hydrochloride buffer (pH 2.7)containing 0.5 M NaCl. About 2.5 ml of a solution, obtained by combiningthe fractions (fractions 3 to 7) in which the IgG protein eluted withthe glycine hydrochloride buffer was detected for each lot, was appliedto a PD-10 desalting column (manufactured by Amersham Biosciences, Inc.)which had previously been equilibrated with Otsuka Physiological Salinefor Injection (TO-SS) containing 0.01% Tween 20, followed by elutionwith TO-SS, whereby 3.5 ml of a sample whose solvent was replaced withTO-SS was obtained. With respect to the IgG protein fractions (fractions16 to 19) eluted with the sodium citrate buffer in an amount of about2.5 ml, the fractions for all the three lots were combined and theresulting solution was concentrated to 2.5 ml with a centrifugalmembrane concentrator Amicon Ultra-4 (manufactured by Millipore Co.,Ltd.). Then, the concentrate was applied to a PD-10 desalting column(manufactured by Amersham Biosciences, Inc.) which was previouslyequilibrated with TO-SS, followed by elution with TO-SS, whereby 3.5 mlof a sample (Citrate-E) whose solvent was replaced with TO-SS wasobtained. The thus prepared samples were cryopreserved at −80° C. untiluse.

b) Measurement of Protein Concentration of Affinity-Purified Anti-MouseSiglec-15 Polyclonal Antibody

For the purified anti-mouse Siglec-15 polyclonal antibody samples (theprotein fractions eluted from the PD-10 desalting column), the proteinconcentration was measured with a DC-Protein Assay kit (manufactured byBio-Rad Laboratories, Inc.) using bovine IgG as a standard sample. Thesamples in which the protein concentration was measured were preparedsuch that the antibody concentration was 15 or 50 μg/ml, and subjectedto electrophoresis and silver staining in the same manner as in b) ofExample 11. The results are shown in FIG. 13. It was shown that an IgGprotein composed of a heavy chain having a molecular weight of about 45kDa and a light chain having a molecular weight of about 21 kDa wasefficiently purified and concentrated in the protein fractions elutedfrom the PD-10 column. As shown in Table 4, about 2.3 to 7.4 mg of anaffinity-purified anti-mouse Siglec-15 polyclonal antibody could beprepared in each of lot Nos. 1 to 3 or as Citrate-E fraction.

TABLE 4 Protein Conc. (mg/ml) Sample Vol. (ml) Total protein (mg) No. 10.661 3.5 2.31 No. 2 1.715 3.5 6.00 No. 3 2.112 3.5 7.39 Citrate- 1.1553.5 4.04 E

Example 15 Purification of Affinity-Purified Anti-Mouse Siglec-15Polyclonal Antibody with Gel Filtration Column a) Superose 6 ColumnChromatography

In order to completely remove endotoxin and low molecular weightimpurities from the affinity-purified anti-mouse Siglec-15 polyclonalantibody prepared in Example 14, purification was further performed witha gel filtration column. 1 ml of each of the affinity-purifiedanti-mouse Siglec-15 polyclonal antibodies Nos. 2 and 3 was applied to aSuperose 6 HR 10/30 column (manufactured by Amersham Biosciences, Inc.)which was previously treated with a pyrogen removing agent PyroCLEAN(manufactured by ALerCHEK, Inc.) and equilibrated with Dulbecco's PBS(D-PBS, manufactured by Invitrogen, Inc.) containing 0.01% Tween 20,followed by elution with D-PBS containing 0.01% Tween 20 at a flow rateof 0.4 ml/min. The chromatograms thereof are shown in FIG. 14 a and FIG.14 b. The eluate was fractionated at 0.5 ml per fraction into mini-sorptubes (manufactured by Nunc, Inc.), and 2.0 ml of a gelfiltration-purified anti-mouse Siglec-15 polyclonal antibody sample(fractions 28 to 31) was obtained. The thus prepared sample wascryopreserved at −80° C. until use.

b) Measurement of Protein Concentration of Rabbit IgG Purified with GelFiltration Column

Also for the gel filtration-purified anti-mouse Siglec-15 polyclonalantibody sample (the protein fractions eluted from the Superose 6column), the protein concentration was measured. As shown in Table 5,the gel filtration-purified anti-mouse Siglec-15 polyclonal antibody inan amount of 2.25 mg and 3.34 mg could be prepared in lot Nos. 2 and 3,respectively.

TABLE 5 Protein Conc. (mg/ml) Sample Vol. (ml) Total protein (mg) No. 20.643 3.5 2.25 No. 3 0.955 3.5 3.34

Example 16 Preparation of Mouse Bone Marrow Nonadherent Cells

The femur and tibia were resected from a male ddY mouse at the age of 5to 8 weeks and soft tissues were removed. Both ends of the femur ortibia were cut off, and D-PBS was injected using a syringe with a25-gauge injection needle to push out bone marrow cells, which werecollected in a centrifugal tube. Centrifugation was performed at roomtemperature for 5 minutes at 100 g, and the supernatant was removed. Tothe cell pellet, 1 ml of a hemolytic buffer (Red Blood Cell LysingBuffer, manufactured by Sigma Co., Ltd.) was added to suspend it, andthe resulting suspension was left at room temperature for 5 minutes. 20ml of D-PBS was added thereto, and the suspension was centrifuged atroom temperature for 5 minutes at 100 g, and the supernatant wasremoved. To the cell pellet, 10 ml of MEM-α medium (manufactured byInvitrogen, Inc.) containing 5 ng/ml of M-CSF (manufactured by R&Dsystems, Inc.) and 10% fetal bovine serum (FBS) was added to suspend it.Then, the resulting suspension was passed through a cell strainer (40 μmNylon, manufactured by BD Falcon) to remove aggregates. The resultingcells were transferred to a 75 cm²-T flask (for the use of adherentcells) and cultured overnight in a CO₂ incubator. After the overnightculture, the cells which did not adhere to the T-flask were recoveredand used as mouse bone marrow nonadherent cells.

Example 17 Effect of Addition of Anti-Mouse Siglec-15 PolyclonalAntibody on Osteoclast Differentiation of Mouse Bone Marrow NonadherentCells (Stimulation with RANKL)

By using the anti-mouse Siglec-15 polyclonal antibodies produced inExamples 14 and 15, an effect on osteoclast differentiation of mousebone marrow nonadherent cells was studied. Mouse bone marrow nonadherentcells prepared by the above-mentioned method in Example 16 were preparedat 1.5×10⁵ cells/ml in α-MEM medium containing 10% FBS and 10 ng/ml ofM-CSF (manufactured by R&D systems, Inc.), and the resulting cellpreparation was seeded in each well of a 96-well plate in an amount of200 μl and the cells were cultured for 2 days in a CO₂ incubator. Theold culture solution in the 96-well plate was removed, and 100 μl ofMEM-α medium containing 10% FBS to which human RANKL (RANKL,manufactured by Peprotech, Inc.) and M-CSF were added to give finalconcentrations of 20 ng/ml and 10 ng/ml, respectively, was added to eachwell. To the cell culture solution, the affinity-purified No. 3antibody, Citrate-E antibody, gel filtration-purified No. 2 antibody,gel filtration-purified No. 3 antibody, pre-immune rabbit IgG (producedin Examples 12, 14, and 15), or commercially available rabbit controlIgG (Non-immune Rabbit IgG CLRB00, manufactured by CedarlaneLaboratories Ltd.) was added at a concentration of from 30 to 1,000ng/ml, and the cells were cultured for an additional 3 days in a CO₂incubator. After completion of the culturing, the activity oftartrate-resistant acid phosphatase (TRAP) of the formed osteoclasts wasmeasured by the following procedure. The culture solution in each wellof the 96-well plate was removed by suction, and 50 μl of 50 mM sodiumcitrate buffer (pH 6.1) containing 1% Triton X-100 was added to eachwell. Then, the plate was shaken for 5 minutes on a plate shaker to lysethe cells. To each well, 50 μl of a substrate solution (50 mM sodiumcitrate buffer (pH 6.1) containing 5 mg/ml p-nitrophenyl phosphate and0.46% sodium tartrate) was added, and the plate was incubated at roomtemperature for 5 minutes. After the incubation, 50 μl of a 1 N sodiumhydroxide solution was added to each well of the 96-well plate to stopthe enzymatic reaction. After stopping the enzymatic reaction, anabsorbance of each well at 405 nm was measured, and the measurement wasused as an index of TRAP activity. The results are shown in FIGS. 15 and16. A significant inhibition of TRAP activity was not observed in thecases of the pre-immune rabbit IgG and the commercially available rabbitcontrol IgG. On the other hand, a significant inhibition of TRAPactivity was observed in the cases of the affinity-purified No. 3antibody at 30 ng/ml or higher and the Citrate-E antibody at about 130ng/ml or higher (FIG. 15). Also in the case of the gelfiltration-purified No. 3 antibody, a significant inhibition of TRAPactivity was observed at 30 ng/ml or higher. Moreover, a more potentinhibitory activity was observed in the case of the gelfiltration-purified No. 3 antibody than in the case of the gelfiltration-purified No. 2 antibody (FIG. 16). Since the activity ofinhibiting osteoclast formation was observed also in the gelfiltration-purified antibody, it was shown that the activity ofinhibiting osteoclast formation observed in the anti-mouse Siglec-15polyclonal antibody is not attributed to endotoxin or low molecularweight impurities contained in the antibody sample, but is attributed tothe activity of the antibody molecule itself. From the above results, itwas shown that the anti-mouse Siglec-15 polyclonal antibody has a potentinhibitory effect on osteoclast formation (osteoclast differentiationand maturation).

Example 18 Neutralization by Antigen of Inhibition of OsteoclastDifferentiation of Mouse Bone Marrow Nonadherent Cells by Addition ofAnti-Mouse Siglec-15 Polyclonal Antibody (Stimulation with RANKL)

It was confirmed that the effect of the anti-mouse Siglec-15 polyclonalantibody depends on the binding thereof to an antigen by previouslyadding the antigen to the anti-mouse Siglec-15 polyclonal antibody toform an immune precipitate. To 10 μg/ml of the affinity-purified No. 3antibody produced in Example 14, the mouse Siglec-15-His or Siglec-15-Fcprepared in Example 8 or 9 was added at a concentration of 10, 30, 100or 300 μg/ml, and the resulting mixture was incubated at 37° C. for 2hours. After the incubation, the mixture was centrifuged for 5 minutesby Chibitan, and the resulting supernatant was sterilized by filtrationthrough a Millex-GV filter (manufactured by Millipore Co., Ltd.). Mousebone marrow nonadherent cells prepared by the method in Example 16 wereseeded in a 96-well plate at 200 μl/well, and the cells were culturedfor 2 days in a CO₂ incubator. The old culture solution in the 96-wellplate was removed, and 100 μl of MEM-α medium containing 10% FBS towhich human RANKL (RANKL, manufactured by Peprotech, Inc.) and M-CSFwere added to give final concentrations of 20 ng/ml and 10 ng/ml,respectively, was added to each well. To the cell culture solution, eachof the test samples prepared in the above was added at 1/200 (v/v), andthe cells were cultured for an additional 3 days in a CO₂ incubator.After completion of the culturing, the activity of tartrate-resistantacid phosphatase (TRAP) of the formed osteoclasts was measured by themethod described in Example 17. The results are shown in FIG. 17. Inboth cases where the antibody was neutralized by Siglec-15-His and wherethe antibody was neutralized by Siglec-15-Fc, the effect of the antibodywas neutralized and lost. These results demonstrated that the inhibitoryeffect of the anti-mouse Siglec-15 polyclonal antibody on osteoclastformation is due to binding thereof to Siglec-15 protein and blocking ofits function.

Example 19 Effect of Addition of Anti-Mouse Siglec-15 PolyclonalAntibody on Osteoclast Differentiation of Mouse Bone Marrow NonadherentCells (Stimulation with TNF)

By using the anti-mouse Siglec-15 polyclonal antibody, an effect onosteoclast differentiation of mouse bone marrow nonadherent cells bystimulation with TNF was studied. Mouse bone marrow nonadherent cellsprepared by the method in Example 16 were prepared at 1.5×10⁵ cells/mlin α-MEM medium containing 10% fetal bovine serum (FBS), 10 ng/ml ofM-CSF and 2 ng/ml of TGF-β (manufactured by R&D systems, Inc.), and theresulting cell preparation was seeded in each well of a 96-well plate inan amount of 200 μl and the cells were cultured for 2 days in a CO₂incubator. The old culture solution in the 96-well plate was removed,and 100 μl of MEM-α medium containing 10% FBS to which human recombinantTNF-α (manufactured by R&D systems, Inc.) and M-CSF were added to givefinal concentrations of 30 ng/ml and 10 ng/ml, respectively, was addedto each well. To the cell culture solution, the pre-immune IgG producedin Example 12 or the gel filtration-purified anti-mouse Siglec-15 No. 3antibody produced in Example 15 was added at a concentration of from 30to 1,000 ng/ml, and the cells were cultured for an additional 3 days ina CO₂ incubator. At the same time, a well in which the cells werecultured by adding human recombinant OCIF/OPG prepared by the methoddescribed in the description of Patent No. WO 96/26217 at aconcentration of from 3 to 100 ng/ml was also prepared. After completionof the culturing, the activity of tartrate-resistant acid phosphatase(TRAP) of the formed osteoclasts was measured by the method described inExample 17. The results are shown in FIG. 18. In the cases of thepre-immune IgG and OCIF/OPG, a significant inhibition of TRAP activitywas not observed. On the other hand, in the case of the gelfiltration-purified anti-mouse Siglec-15 No. 3 antibody, about 50%inhibition of TRAP activity was observed at a concentration of 250 ng/mlor higher. From these results, it was shown that the anti-mouseSiglec-15 polyclonal antibody can inhibit also TNF-induced osteoclastformation (osteoclast differentiation and maturation) which cannot beinhibited by OCIF/OPG.

For each well of a 96-well plate prepared by performing culturing in thesame manner as described above, TRAP staining was performed using aLeukocyte Acid Phosphatase kit (manufactured by Sigma Co., Ltd.)according to the protocol attached to the kit, and the formation ofTRAP-positive multinucleated osteoclasts was observed. As a result, theformation of TRAP-positive giant multinucleated osteoclasts wasinhibited by the addition of the anti-mouse Siglec-15 polyclonalantibody (FIG. 19). Since mononuclear osteoclasts were formed even inthe case where the anti-mouse Siglec-15 polyclonal antibody was added,it was shown that the anti-mouse Siglec-15 polyclonal antibody stronglyinhibits the process of cell fusion in osteoclast differentiation andmaturation induced by TNF.

Example 20 Effect of Addition of Anti-Mouse Siglec-15 PolyclonalAntibody on Osteoclast Differentiation of Mouse Bone Marrow-DerivedPrimary Cultured Cells (TRAP Activity)

A male ddY mouse at the age of 7 weeks was euthanized by cervicaldislocation under ether anesthesia and the femur and tibia wereresected. After soft tissues were removed, both ends of the femur ortibia were cut off. Then, α-MEM medium containing 10% fetal bovine serumwas injected into the bone marrow using a syringe barrel with a 25-gaugeinjection needle, and bone marrow cells were collected. After the numberof cells was counted, the cells were prepared at 5×10⁶ cells/ml in α-MEMmedium containing 10% fetal bovine serum. The resulting cell preparationwas plated in a 96-well plate at 100 μl/well, and active vitamin D₃(manufactured by Sigma Co., Ltd.) was added thereto to give a finalconcentration of 2×10⁻⁸ M. To this cell culture supernatant, theaffinity-purified anti-mouse Siglec-15 No. 3 antibody produced inExample 14 or the pre-immune rabbit IgG produced in Example 12 was addedto give a final concentration of 4.57, 13.7, 41.2, 123, 370, 1,111,3,333, or 10,000 ng/ml, and the cells were cultured for 8 days in a CO₂incubator. Incidentally, the medium replacement and addition of a testsubstance were performed on days 3 and 6. The culture supernatant wasremoved on day 8 of culture, and 10% neutral formalin was added to fixthe cells. After fixing the cells, the cells were washed twice withdistilled water, and a TRAP substrate solution (15 mM p-nitrophenylphosphate, 50 mM sodium tartrate, 0.1 M sodium acetate buffer (pH 5.0))was added thereto at 100 μl/well, and a reaction was allowed to proceedat room temperature for 30 minutes. Then, 1 N NaOH was added thereto at50 μl/well to stop the reaction, and an absorbance at 405 nm wasmeasured using a microplate reader, whereby the TRAP activity in thecells was evaluated. As a result, the TRAP activity was inhibiteddepending on the dose of the added anti-mouse Siglec-15 polyclonalantibody (FIG. 20-A). On the other hand, in the case where thepre-immune IgG was added, a decrease in the TRAP activity was notobserved (FIG. 20-B) In this manner, it was revealed that the formationof TRAP-positive osteoclasts from mouse bone marrow cells induced byactive vitamin D₃ is inhibited by the antibody specifically binding toSiglec-15.

Example 21 Effect of Addition of Anti-Mouse Siglec-15 PolyclonalAntibody on Cell Fusion of Osteoclasts from Mouse Bone Marrow-DerivedPrimary Cultured Cells (TRAP Staining)

A male ddY mouse at the age of 7 weeks was euthanized by cervicaldislocation under ether anesthesia and the femur and tibia wereresected. After soft tissues were removed, both ends of the femur ortibia were cut off. Then, α-MEM medium containing 10% fetal bovine serumwas injected into the bone marrow using a syringe barrel with a 25-gaugeinjection needle, and bone marrow cells were collected. After the numberof cells was counted, the cells were prepared at 5×10⁶ cells/ml in α-MEMmedium containing 10% fetal bovine serum. The resulting cell preparationwas plated in a 96-well plate at 100 μl/well. By adding active vitaminD₃ (manufactured by Sigma Co., Ltd.) at a final concentration of 2×10⁻⁸M or human RANKL (manufactured by PeproTech Inc.) at a finalconcentration of 80 ng/ml to this culture supernatant as an osteoclastdifferentiation-inducing factor, cell culture supernatants wereprepared. To each of these cell culture supernatants, theaffinity-purified anti-mouse Siglec-15 No. 3 antibody produced inExample 14 was added to give a final concentration of 370 or 3,333ng/ml, or the pre-immune rabbit IgG produced in Example 12 was added togive a final concentration of 3,333 ng/ml. In the culture system ofinducing osteoclast differentiation by active vitamin D₃, the cells werecultured for 8 days in a CO₂ incubator, and in the system of inducingthe differentiation by human RANKL, the cells were cultured for 6 daysin a CO₂ incubator. Incidentally, the medium replacement and addition ofa test substance were performed on days 3 and 6. After the culturing,the supernatant was removed, and 10% neutral formalin was added to fixthe cells. After fixing the cells, the cells were washed twice withdistilled water, and a TRAP staining solution (0.27 mM naphthol AS-MXphosphate (manufactured by Sigma Co., Ltd.), 1.6 mM fast red violet LBsalt (manufactured by Sigma Co., Ltd.), 1% dimethylformamide, 50 mMsodium tartrate, 0.1 M sodium acetate buffer (pH 5.0)) was added at 100μl/well, and a reaction was allowed to proceed at room temperature for 5minutes. Then, the cells were washed twice with distilled water, andthen, observed by microscopy. As a result, in the case where osteoclastdifferentiation was induced by either active vitamin D₃ (FIG. 21) orhuman RANKL (FIG. 22), cell fusion of osteoclasts was inhibited by theaddition of the anti-mouse Siglec-15 polyclonal antibody, and theformation of giant osteoclasts was not observed. On the other hand, inthe case where the pre-immune IgG was added, such inhibition of cellfusion of osteoclasts was not observed. In this manner, it was revealedthat multinucleation and cell fusion of TRAP-positive osteoclasts frommouse bone marrow cells induced by active vitamin D₃ or human RANKL areinhibited by the antibody specifically binding to Siglec-15.

Example 22 Effect of Addition of Anti-Mouse Siglec-15 PolyclonalAntibody on Cell Fusion of Osteoclasts from RAW 264.7 Cells (TRAPStaining) a) Production of Antigenic Protein-Absorbed Anti-MouseSiglec-15 Polyclonal Antibody

Five mixed solutions (A to E) were prepared using D-PBS (manufactured byInvitrogen, Inc.) containing 0.01% Tween 20, such that the respectiveconcentrations of the affinity-purified anti-mouse Siglec-15 No. 3antibody produced in Example 14, the mouse Siglec-15-His protein and themouse Siglec-15-Fc protein produced in Examples 8 and 9 were as shown inTable 6. These mixed solutions were incubated at 37° C. for 2 hours andcentrifuged at 20,000×g for 10 minutes. The resulting supernatants wereused as 20-fold concentration test samples.

TABLE 6 A B C D E anti-Siglec-15 antibody 20 20 20 20 20 Siglec-15-Fc 020 200 0 0 Siglec-15-His 0 0 0 20 200 (Unit: μg/ml)

b) Evaluation Using RAW 264.7 by TRAP Staining

RAW 264.7 was prepared at 2.25×10⁴ cells/ml in α-MEM medium containing10% fetal bovine serum, and the resulting cell preparation was plated ina 96-well plate at 200 μl/well, and human RANKL (manufactured byPeproTech Inc.) was added thereto to give a final concentration of 40ng/ml. To this cell culture supernatant, each of the test samples A to Eproduced in a) was added at a final concentration of 1/20, and the cellswere cultured for 3 days in a CO₂ incubator. After the culturing, thesupernatant was removed, and 10% neutral formalin was added to fix thecells. After fixing the cells, the cells were washed twice withdistilled water, and a TRAP staining solution (0.27 mM naphthol AS-MXphosphate (manufactured by Sigma Co., Ltd.), 1.6 mM fast red violet LBsalt (manufactured by Sigma Co., Ltd.), 1% dimethylformamide, 50 mMsodium tartrate, 0.1 M sodium acetate buffer (pH 5.0)) was added at 100μl/well, and a reaction was allowed to proceed at room temperature for 5minutes. Then, the cells were washed twice with distilled water, andthen, observed by microscopy. As a result, by adding the anti-mouseSiglec-15 polyclonal antibody, cell fusion of osteoclasts wassignificantly inhibited (FIG. 23-A) as compared with the case of thecontrol (FIG. 23-0) without the addition of the test sample. However, byabsorbing the anti-mouse Siglec-15 polyclonal antibody in the mouseSiglec-15-Fc protein used as the immunizing antigen, the inhibitoryeffect of the anti-mouse Siglec-15 polyclonal antibody on cell fusion ofosteoclasts was cancelled (FIGS. 23-B,C). Further, also by absorbing theanti-mouse Siglec-15 polyclonal antibody in the mouse Siglec-15-Hisprotein, the inhibitory effect of the anti-mouse Siglec-15 polyclonalantibody on cell fusion of osteoclasts was cancelled in the same manner(FIGS. 23-D, E). From these results, it was revealed thatmultinucleation and cell fusion of TRAP-positive osteoclasts from RAW264.7 cells induced by human RANKL are inhibited by the antibodyspecifically binding to Siglec-15. Incidentally, the results representedby the symbols A to E in FIG. 23 correspond to the test samples A to Ein Table 6, respectively.

Example 23 Establishment of Rat Anti-Mouse Siglec-15 MonoclonalAntibody-Producing Hybridoma a) Preparation of Antigen

The mouse Siglec-15-His protein produced in Example 8 was prepared at100 μg/0.5 ml, and an equivalent amount of an adjuvant was added theretoand an emulsion was produced using a glass syringe. As the adjuvant,Freund's complete adjuvant (FCA, Manufactured by Difco Laboratories,Inc.) was used only for the first immunization, and Freund's incompleteadjuvant (FICA, Manufactured by Difco Laboratories, Inc.) was used forthe second and subsequent immunizations.

b) Immunization of Rat

Four rats (Wistar, female, 6 weeks of age, purchased from CLEA Japan,Inc.) were used as immunized animals. The emulsion obtained in a) wasinjected subcutaneously and intradermally using a 27 G injection needlesuch that the amount of the antigen was 50 μg per rat. Immunization wasperformed a total of 4 times every 7 days after the first immunization.A small amount (200 μl) of the blood was collected from the tail veinafter 7 days from the date of the 4th immunization, and an antiserum wasprepared. In order to confirm the antibody titer of the antiserum, ELISAusing immobilized mouse Siglec-15-His protein used as the antigen, themouse Siglec-15-Fc protein produced in Example 9, or bovine serumalbumin (BSA) was performed. As a result, the reactivity with the mouseSiglec-15-His protein and mouse Siglec-15-Fc protein was observed in allfour rats (rat Nos. 1 to 4). On the other hand, the reactivity with BSAwas not observed. From these results, it was confirmed that the antibodytiter in the serum of each of the immunized rats increased, andtherefore, the No. 2 rat which showed the highest antibody titer wassubjected to a cell fusion procedure.

c) Cell Fusion

Cell fusion was performed according to a common method of fusing mouse(rat) spleen cells with myeloma cells. The whole blood was collectedfrom the heart of the rat under ether anesthesia and the rat waseuthanized, and then, the spleen was resected. The collected spleencells and P3X63Ag8.653 cells (ATCC CRL 1580) which are mouse myelomacells were subjected to cell fusion using polyethylene glycol (PEG). Theresulting cells were seeded in a 96-well plate, and a medium containinghypoxanthine (H), aminopterin (A) and thymidine (T) (HAT selectionmedium) was added thereto, and then, the cells were cultured for 7 to 10days. The culture supernatant was collected from 61 wells in which thesurvival of hybridomas obtained by cell fusion was confirmed. Then, theantibody titer was evaluated by ELISA using immobilized mouseSiglec-15-His protein, mouse Siglec-15-Fc protein produced in Example 9,or BSA as the antigen, and anti-mouse Siglec-15 monoclonalantibody-producing hybridomas were screened. From the results of thescreening, 12 wells showing a high antibody titer were selected and thehybridomas contained in the wells were subjected to a cloning procedure.

d) Cloning of Hybridoma

For the thus selected hybridomas, first cloning was performed by alimiting dilution method. After limiting dilution, the hybridomas werecultured for 2 weeks, and the antibody titer in the culture supernatantwas confirmed by ELISA using immobilized mouse Siglec-15-Fc proteinproduced in Example 9 or BSA. For 11 clones which were confirmed to bepositive clones, second cloning was performed (in the same manner as thefirst cloning), whereby 10 clones of the anti-mouse Siglec-15 monoclonalantibody-producing hybridomas (#1A1, #3A1, #8A1, #24A1, #32A1, #34A1,#39A1, #40A1, #41B1, #61A1) were established in the end. Incidentally,the hybridomas #32A1 and #41B1 were deposited at the InternationalPatent Organism Depositary of the National Institute of AdvancedIndustrial Science and Technology (located at Central 6, 1-1-1 Higashi,Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Aug. 28, 2008. Thehybridoma #32A1 has been given a deposit number of FERM BP-10999 underthe name of anti-Siglec-15 Hybridoma #32A1, and the hybridoma #41B1 hasbeen given a deposit number of FERM BP-11000 under the name ofanti-Siglec-15 Hybridoma #41B1.

Example 24 Preparation of Rat Anti-Mouse Siglec-15 Monoclonal Antibodya) Preparation of Nude Mouse Ascites

The hybridomas established in Example 23 were cultured using TIL Media I(manufactured by Immuno-biological Laboratories Co., Ltd.) mediumcontaining 10% FCS. Subculturing of the cells was carried out byperforming a procedure in which the culture solution was diluted toabout one-fourth every two to three days by using the time point whenthe cells were grown to about 5×10⁵ cells/ml as a guide. Each thuscultured hybridoma was intraperitoneally implanted in a nude mouse towhich pristane had previously been intraperitoneally administered (0.2ml/mouse) at 1×10⁷ cells per mouse. In the implantation, three nude micewere used for each of the 10 clones of hybridomas. After theimplantation, the ascites was collected at the time when sufficientaccumulation of ascites was observed, which was combined with thosecollected from the other two mice implanted with the same hybridoma andthe amount of the ascites thus combined was measured, and the asciteswas cryopreserved until purification of the antibody. The amounts of thecollected ascites for the respective hybridomas were summarized in Table7.

TABLE 7 Amount of collected ascites Hybridoma (ml) #1A1 12.5 #3A1 8.0#8A1 6.0 #24A1 7.8 #32A1 5.5 #34A1 8.2 #39A1 14.5 #40A1 20.3 #41B1 10.5#61A1 12.3

b) Purification of Antibody

The total amount of the collected ascites was subjected to IgGpurification using a 20 ml Protein G column (manufactured by GEHealthcare, Co., Ltd.). The purified IgG was assayed for purity by a gelfiltration analysis (Superdex 200 column chromatography), and some ofthe antibodies were subjected to centrifugal membrane concentration.That is, 9 types of antibodies except for the #24A1 antibody wereconcentrated to about one-sixth to one-eighth of the original volume bycentrifuging the antibodies at 3,000 rpm for 30 to 60 minutes at 4° C.using a centrifugal membrane concentrator Amicon Ultra-15 (manufacturedby Millipore Co., Ltd.). Subsequently, for the #24A1 antibody and theother concentrated 9 types of antibodies, the protein concentration wasmeasured with a DC-Protein Assay kit (manufactured by Bio-RadLaboratories, Inc.) using bovine serum albumin (BSA) as a standardsample. By the above-mentioned procedure, the anti-mouse Siglec-15monoclonal antibody was prepared.

Example 25 Evaluation of Binding Property of Rat Anti-Mouse Siglec-15Monoclonal Antibody to Mouse Siglec-15 Protein

The binding property of the rat anti-mouse Siglec-15 monoclonal antibodyto mouse Siglec-15 protein was evaluated by an ELISA method. The mouseSiglec-15-Fc protein produced in Example 9 was diluted to 5 μg/ml with0.1 M sodium carbonate buffer (pH 9.5), and the resulting solution wasadded to a 96-well plate (manufactured by Nalge Nunc International,Inc., Cat. No. 430341) at 100 μl/well. After the plate was left at roomtemperature for 1 hour, the solution was removed and a washing buffer(phosphate-buffered saline containing 0.05% Tween 20) was added at 300μl/well and removed. After this washing procedure was performed one moretime, phosphate-buffered saline containing 25% BlockAce (manufactured byDainippon Sumitomo Pharma Co., Ltd.) was added at 200 μl/well, and theplate was left at room temperature for 1 hour, whereby blocking waseffected. The liquid was removed, and the plate was washed twice with300 μl/well of washing buffer. Then, each of the rat anti-mouseSiglec-15 monoclonal antibodies prepared in Example 24 or rat controlIgG (manufactured by R&D systems, Inc.) was diluted to a finalconcentration of from 1.28 to 20,000 ng/ml (5-fold dilution series) withan ELISA buffer (phosphate-buffered saline containing 12.5% BlockAce and0.05% Tween 20), and the resulting diluted antibody solution was addedto the plate at 100 μl/well. After the plate was left at roomtemperature for 1 hour, the liquid was removed, and the plate was washedthree times with 300 μl/well of washing buffer. Subsequently, HRP(horseradish peroxidase)-labeled goat anti-rat IgG antibody(manufactured by Beckman Coulter, Inc.) diluted to 1,000-fold with theELISA buffer was added at 100 μl/well, and the plate was left at roomtemperature for 1 hour. The liquid was removed and the plate was washedthree times with 300 μl/well of washing buffer, and then, by using acolor developing kit for peroxidase (manufactured by Sumitomo BakeliteCo., Ltd.), the color was developed according to the protocol attachedto the kit. After developing the color, the absorbance at 492 nm wasmeasured using a microplate reader (manufactured by Molecular DevicesCorporation, Japan). As a result, it was confirmed that all the 10 testsubstances of the rat anti-mouse Siglec-15 monoclonal antibodiesexamined bind to the mouse Siglec-15 protein in an antibodyconcentration-dependent manner (FIG. 24). On the other hand, in the caseof the rat control IgG, binding to the mouse Siglec-15 protein was notobserved.

Example 26 Evaluation of Biological Activity of Rat Anti-Mouse Siglec-15Monoclonal Antibody Based on Test for Mouse Osteoclast Formation

By using all the 10 test substances of the anti-mouse Siglec-15monoclonal antibodies produced in Example 24, an effect on osteoclastdifferentiation of mouse bone marrow nonadherent cells was examined.Mouse bone marrow nonadherent cells prepared by the method in Example 16were prepared at 1.5×10⁵ cells/ml in α-MEM medium containing 10% FBS and10 ng/ml of M-CSF (manufactured by R&D systems, Inc.), and the resultingcell preparation was seeded in each well of a 96-well plate in an amountof 200 μl and the cells were cultured for 2 days in a CO₂ incubator. Theold culture solution in the 96-well plate was removed, and 100 μl ofMEM-α medium containing 10% FBS to which human RANKL (RANKL,manufactured by Peprotech, Inc.) and M-CSF were added to give finalconcentrations of 20 ng/ml and 10 ng/ml, respectively, was added to eachwell. To the cell culture solution, each of the rat anti-mouse Siglec-15monoclonal antibodies produced in Example 24, a sample obtained byremoving sodium azide from commercially available rat control IgG(purified rat IgG, manufactured by R&D systems, Inc.) or the rabbitanti-mouse Siglec-15 polyclonal antibody (No. 3) produced in Example 14was added at a concentration of from 32 to 1,000 ng/ml, and the cellswere cultured for an additional 3 days in a CO₂ incubator. Aftercompletion of the culturing, the activity of tartrate-resistant acidphosphatase (TRAP) of the formed osteoclasts was measured by the methoddescribed in Example 17. After stopping the enzymatic reaction, theabsorbance of each well at 405 nm was measured, and the measurement wasused as an index of TRAP activity. The results are shown in FIGS. 25 and26. A significant inhibition of TRAP activity was not observed in thecase of the commercially available rat control IgG. On the other hand, asignificant inhibition of TRAP activity was observed in the cases of the#32A1 antibody in the range of from 32 ng/ml to 1000 ng/ml, the #8A1antibody and the affinity-purified rabbit polyclonal No. 3 antibody inthe range of from 63 ng/ml to 1000 ng/ml. Also in the cases of the #3A1antibody, #34A1 antibody, and #39A1 antibody, a dose-dependentinhibition of TRAP activity was observed at a relatively higherconcentration of 500 ng/ml or higher. The inhibition of mouse osteoclastformation by the other antibodies was not observed. From the aboveresults, antibodies which strongly inhibit mouse osteoclast formation(osteoclast differentiation and maturation) were found among theprepared rat anti-mouse Siglec-15 monoclonal antibodies. Further, as aproperty common to the #3A1 antibody, #8A1 antibody, #32A1 antibody,#34A1 antibody, and #39A1 antibody, an activity of inhibiting osteoclastformation at a concentration of 1000 ng/ml, i.e., 1 μg/ml or less can beexemplified.

Example 27 Extraction of Total RNA from Human-Derived Mature Osteoclasts

When normal human osteoclast precursor cells (Normal Human NaturalOsteoclast Precursor Cells, purchased from Sanko Junyaku Co., Ltd., Cat.No. 2T-110) are cultured in a minimal essential medium for osteoclastprecursor cells (OPBM, purchased from Sanko Junyaku Co., Ltd., Cat. No.PT-8201) supplemented with an OPGM supplement set (OsteoclastSingleQuot™ Kit, purchased from Sanko Junyaku Co., Ltd., Cat. No.PT-9501) containing fetal bovine serum (final concentration: 10%), humanRANKL, human M-CSF and the like, a large number of TRAP-positivemultinucleated osteoclasts arise after 3 to 7 days. By using this cellculturing system according to the protocol attached to the kit,human-derived mature osteoclasts were produced.

a) The normal human osteoclast precursor cells were seeded in 60 wellsof a 96-well plate at 1×10⁴ cells/well, and human RANKL was addedthereto to give a final concentration of 66 ng/ml, and the cells werecultured for 4 days in a CO₂ incubator. Then, the total RNA wasextracted from the multinucleated osteoclasts using a total RNAextraction reagent (ISOGEN, manufactured by Nippon Gene Co., Ltd.)according to the protocol attached to the reagent. The collected totalRNA was stored at −80° C. until use.

b) The normal human osteoclast precursor cells were seeded in 84 wellsof each of two 96-well plates at 1×10⁴ cells/well, and human RANKL wasadded to each well of one of the plates to give a final concentration of53.2 ng/ml, and human RANKL was not added to the other plate, and thecells were cultured for 3 days in a CO₂ incubator. Then, the total RNAwas extracted from the cells using a total RNA extraction reagent(ISOGEN, manufactured by Nippon Gene Co., Ltd.) according to theprotocol attached to the reagent. The collected total RNA was stored at−80° C. until use.

Example 28 Acquisition of Sequence of Open Reading Frame (ORF) for HumanSiglec-15

a) Synthesis of First Strand cDNA

By using the total RNA produced in a) of Example 27 as a template,synthesis of cDNA was performed using oligo(dT) primer (manufactured byInvitrogen, Inc.) and Superscript III reverse transcriptase(manufactured by Invitrogen, Inc.). The procedure was performedaccording to the protocol attached to the enzyme.

b) PCR Reaction

Oligonucleotides having the sequences of: 5′-attaagcttc accATGGAAAAGTCCATCTG GCTGC-3′ (hSiglec-15-HindIII kozak-F: SEQ ID NO: 29 in theSequence Listing); and 5′-agtggatccT CACGGTGAGC ACATGGTGGC-3′(hSiglec-15-BamHI-R: SEQ ID NO: 30 in the Sequence Listing) as primersfor amplifying the ORF cDNA for human Siglec-15 by PCR were synthesizedaccording to a common procedure. The PCR was performed using thiscombination of primers and the cDNA produced in a) according to a commonprocedure. The resulting PCR reaction solution was purified usingPureLink PCR Purification Kit (manufactured by Invitrogen, Inc.).

c) Cloning into pcDNA3.1(+) Vector

The purified PCR reaction solution obtained in b) and pcDNA3.1(+) vector(manufactured by Invitrogen, Inc.) were treated with restriction enzymes(BamHI, HindIII), followed by gel cutting and purification, and then, aligase reaction was performed according to a common procedure.Escherichia coli TOP10 was transformed, and colony PCR was performed forthe resulting drug-resistant clones. The entire nucleotide sequence ofthe ORF cDNA inserted into a plasmid was analyzed using a DNA sequencerfor a clone in which an amplified product with a predicted size wasobtained, and as a result, it was found to be the sequence representedby SEQ ID NO: 1 in the Sequence Listing. This nucleotide sequence wasthe same as the ORF coding region of the sequence registered in NCBIGeneBank database as “human Siglec-15” (accession number: NM 213602),and further, the amino acid sequence (SEQ ID NO: 2 in the SequenceListing) encoded by the nucleotide sequence was 100% identical to theamino acid sequence of human Siglec-15.

Example 29 Expression of mRNA for Human Siglec-15 Accompanying HumanOsteoclast Differentiation (Real-Time PCR Analysis)

To 1 μg of the total RNA produced in b) of Example 27, 1 μl of 1 U/μlDNase I and 1 μl of 10× DNase I buffer (manufactured by Invitrogen,Inc.) were added, and then, the final volume was brought to 10 μl withH₂O. After a reaction was allowed to proceed at room temperature for 15minutes, 1 μl of 25 mM EDTA was added thereto and the resulting mixturewas heated at 65° C. for 10 minutes. From this solution, an 8 μl aliquotwas taken, and 1 μl of 50 μM oligo(dT)₂₀ primer and 1 μl of 10 mM dNTPswere added thereto, and the resulting mixture was heated at 65° C. for 5minutes and then incubated in ice. To this solution, 2 μl of 10×RTbuffer (manufactured by Invitrogen, Inc.), 4 μl of 25 mM MgCl₂, 2 μl of0.1 M dithiothreitol, 1 μl of RNase inhibitor (RNaseOUT, 40 U/μl,manufactured by Invitrogen, Inc.), and 1 μl of Superscript III reversetranscriptase (200 U/μl, manufactured by Invitrogen, Inc.) were addedand the total volume was brought to 20 μl. After a reaction was allowedto proceed at 50° C. for 50 minutes, the mixture was heated at 85° C.for 5 minutes and then incubated in ice.

By using the thus produced single-stranded cDNA, real-time PCR wasperformed using a combination of the following primers and fluorescentlylabeled probes (TaqMan probe, manufactured by Applied Biosystems, Inc.).

Conditions for Real-Time PCR:

Primers for amplifying human cathepsin K: 5′-ccgcagtaat gacacccttt-3′(TqM-hcatK-F: SEQ ID NO: 31 in the Sequence Listing) and5′-aaggcattgg tcatgtagcc-3′ (TqM-hcatK-R: SEQ ID NO: 32 in the SequenceListing) TaqMan probe for detecting human cathepsin K:5′-Fam-tcagggtcag tgtggttcct gttgggct-TAMRA-3′(TqM-hcatK-probe: SEQ ID NO: 33 in the Sequence Listing)Primers for amplifying human TRAP: 5′-ctgtcctggc tcaagaaaca-3′(TqM-hTRAP-F: SEQ ID NO: 34 in the Sequence Listing) and5′-ccatagtgga agcgcagata-3′ (TqM-hTRAP-R: SEQ ID NO: 35 in the SequenceListing) TaqMan probe for detecting humanTRAP:5′-Fam-tgagaatggc gtgggctacg tgctgagt-TAMRA-3′(TqM-hTRAP-probe: SEQ ID NO: 36 in the Sequence Listing)Primers for amplifying human Siglec-15: 5′-cagccaccaa catccatttc-3′(TqM-hSiglec-15-F: SEQ ID NO: 37 in the Sequence Listing) and5′-cgctcaagct aatgcgtgta-3′ (TqM-hSiglec-15-R: SEQ ID NO: 38 in theSequence Listing) TaqMan probe for detecting human Siglec-155′-Fam-aagaacaaag gccagtgcga ggcttggc-TAMRA-3′(TqM-hSiglec-15-probe: SEQ ID NO: 39 in the Sequence Listing)Primers for amplifying human L32 ribosomal protein:5′-gagatcgctc acaatgtttc ct-3′(TqM-hL32-F: SEQ ID NO: 40 in the Sequence Listing) and5′-gatgccagat ggcagttttt ac-3′(TqM-hL32-R: SEQ ID NO: 41 in the Sequence Listing)TaqMan probe for detecting human L32 ribosomal protein:5′-Fam-accgcaaagc catcgtggaa agagctg-TAMRA-3′(TqM-hL32-probe: SEQ ID NO: 42 in the Sequence Listing)

A real-time PCR analysis was performed using a real-time PCR system (ABIPrism 7700 Sequence Detector, manufactured by Perkin Elmer Japan AppliedBiosystems Division) under the following conditions. In the reaction,TaqMan Universal PCR Master Mix (manufactured by Applied Biosystems,Inc.) was used. First, distilled water was added to 25 pmol of eachprimer, 8 ng of single-stranded cDNA and 10 pmol of TaqMan probe tobring the final volume to 25 μl, and then, 25 μl of TaqMan Universal PCRMaster Mix was added thereto, whereby 50 μl of a reaction solution wasprepared. This reaction solution was heated at 50° C. for 2 minutes andthen heated at 95° C. for 10 minutes, and thereafter subjected to 40temperature cycles of “95° C. for 0.25 minutes and 60° C. for 1 minute”,whereby a real-time PCR analysis was performed. Incidentally, theexpression level of mRNA for each gene was corrected by the expressionlevel of mRNA for L32 ribosomal protein.

As a result, it was revealed that the expression level of the Siglec-15gene significantly increased in the case where human osteoclastdifferentiation was induced by adding RANKL in the same manner ascathepsin K and TRAP genes which are known as marker molecules forosteoclasts (FIG. 27).

Example 30 Production of Soluble Human Siglec-15 Protein ExpressionConstruct

A partial nucleic acid sequence encoding the extracellular domain ofhuman Siglec-15 protein is represented by SEQ ID NO: 43 in the SequenceListing and the amino acid sequence thereof is represented by SEQ ID NO:44 in the Sequence Listing. By utilizing such a partial sequence,soluble human Siglec-15 protein can be produced in a culture supernatantof an animal cell or the like.

a) Amplification of Soluble Human Siglec-15 Gene by PCR

Oligonucleotides having the sequences of: 5′-ggggacaagt ttgtacaaaaaagcaggctt caccATGGAA AAGTCCATCT GGCTGC-3′ (hSiglec-15-ECD-F: SEQ ID NO:45 in the Sequence Listing); and 5′-ggggaccact ttgtacaaga aagctgggtcCCCGCTGGCG CCATGGAAGC GG-3′ (hSiglec-15-ECD-R: SEQ ID NO: 46 in theSequence Listing) as primers for amplifying the human Siglec-15extracellular domain cDNA by PCR were synthesized according to a commonprocedure. Incidentally, these primers were designed, as amplificationprimers for producing a gateway entry clone, such that an attB1 sequenceis added to hSiglec-15-ECD-F and an attB2 sequence is added tohSiglec-15-ECD-R. The PCR was performed using this combination ofprimers and the human Siglec-15/pcDNA3.1(+) plasmid produced in Example28 as a template according to a common procedure. The resulting PCRreaction solution was purified using PureLink PCR Purification Kit(manufactured by Invitrogen, Inc.).

b) Production of Entry Clone by Gateway BP Reaction

An entry clone into which the human Siglec-15 extracellular domain cDNAwas integrated by the Gateway technology (Invitrogen, Inc.) employing alambda phage site-specific recombination system was produced in thefollowing method. First, a BP reaction using BP Clonase was performedbetween the PCR product having an attB sequence at both ends produced ina) and pDNOR221 (manufactured by Invitrogen, Inc.) which is a donorvector having an attP sequence. By using this reaction solution,Escherichia coli TOP10 was transformed, colony PCR was performed fordrug-resistant clones, and the size of inserts was confirmed. Then, fora clone confirmed to have an insert with a correct size, a sequenceanalysis of the total DNA sequence of the insert was performed. As aresult, an entry clone which is completely identical to the targetnucleic acid sequence (SEQ ID NO: 43 in the Sequence Listing) encodingthe extracellular domain of human Siglec-15 protein was obtained.

c) Production of Expression Clone by Gateway LR Reaction

An expression clone into which the human Siglec-15 extracellular domaincDNA was integrated by the Gateway technology (Invitrogen, Inc.)employing a lambda phage site-specific recombination system was producedby the following method. The entry clone produced in b) contains aninsert having an attL sequence at both ends. An LR reaction using LRClonase was performed between this entry clone and two types ofdestination vectors having an attR sequence. Incidentally, as thedestination vectors, two types of destination vectors: pDONM designedsuch that a V5 epitope tag and a 6×His tag are added to the C terminusof the insert; and phIgFc designed such that a human Fc tag is added tothe C terminus of the insert were used. By using the reaction solutionobtained by the LR reaction, Escherichia coli TOP10 was transformed, anda sequence analysis was performed for the resulting drug-resistantclones to confirm whether correct recombination occurred.

As a result of the sequence analysis, expression clones (soluble humanSiglec-15/pDONM and soluble human Siglec-15/phIgFc) in which correctrecombination occurred were obtained for both pDONM and phIgFc,respectively. By transfecting the soluble human Siglec-15/pDONM into ananimal cell or the like, mRNA having the base sequence represented bySEQ ID NO: 47 in the Sequence Listing is transcribed and translated intoa protein (human Siglec-15-His) having the amino acid sequencerepresented by SEQ ID NO: 48 in the Sequence Listing. Further, bytransfecting the soluble human Siglec-15/phIgFc into an animal cell orthe like, mRNA having the base sequence represented by SEQ ID NO: 49 inthe Sequence Listing is transcribed and translated into a protein (humanSiglec-15-Fc) having the amino acid sequence represented by SEQ ID NO:50 in the Sequence Listing.

Example 31 Large-Scale Preparation of Culture Solution ContainingSoluble Human Siglec-15 Protein Using 293-F Cells a) Preparation ofCulture Solution Containing Human Siglec-15-his

The soluble human Siglec-15/pDONM obtained in Example 30 was prepared inan amount of about 25 mg. Incidentally, in the purification of a plasmidfrom Escherichia coli cultured on a large scale, Invitrogen PureLinkHiPure Plasmid Gigaprep Kit (manufactured by Invitrogen, Inc.) was used.The thus prepared plasmid was mixed with Opti-MEM (manufactured byInvitrogen, Inc.), and 50 ml of a transfection reagent 293fectin(manufactured by Invitrogen, Inc.) was added thereto, and the resultingmixture was incubated at room temperature for 20 minutes. This mixturewas added to FreeStyle 293-F cells (manufactured by Invitrogen, Inc.)cultured in FreeStyle 293 Expression Medium (manufactured by Invitrogen,Inc.) containing 1% penicillin-streptomycin such that the cell densityreached 1.0 to 3.4×10⁶ cells/ml using a 25 L bioprocess cultureapparatus (WAVE Bioreactor). After the cells were subjected to spinnerculture (30 rotations/min) at a CO₂ concentration of from 6 to 12% for96 hours (4 days) at 37° C., the culture solution was collected andcentrifuged to prepare a culture supernatant. It is considered that inthe thus prepared culture supernatant, a protein in which a V5 epitopetag and a 6×His tag have been added to the C-terminal side of the humanSiglec-15 extracellular domain (human Siglec-15-His) is expressed.

b) Preparation of Culture Solution Containing Human Siglec-15-Fc

The soluble human Siglec-15/phIgFc obtained in Example 30 was preparedin an amount of about 5 mg. Incidentally, in the purification of plasmidfrom Escherichia coli cultured on a large scale, Invitrogen PureLinkHiPure Plasmid Gigaprep Kit (manufactured by Invitrogen, Inc.) was used.The thus prepared plasmid was mixed with Opti-MEM (manufactured byInvitrogen, Inc.), followed by filter sterilization. Then, 10 ml of atransfection reagent 293fectin (manufactured by Invitrogen, Inc.) wasadded thereto, and the resulting mixture was incubated at roomtemperature for 20 minutes. This mixture was added to FreeStyle 293-Fcells (manufactured by Invitrogen, Inc.) cultured in Erlenmeyer flaskssuch that the cell density reached 1.0 to 3.0×10⁶ cells/ml×5 L (1L/flask×5 flasks) in FreeStyle 293 Expression Medium (manufactured byInvitrogen, Inc.). After the cells were subjected to rotary culture (125rotations/min) at a CO₂ concentration of 8.0% for 96 hours (4 days) at37° C., the culture solution was collected and centrifuged to prepare aculture supernatant. It is considered that in the thus prepared culturesupernatant, a protein in which a human Fc tag has been added to theC-terminal side of the human Siglec-15 extracellular domain (humanSiglec-15-Fc) is expressed.

Example 32 Purification of Soluble Human Siglec-15 Protein a)Purification of Soluble Human Siglec-15-his

a-i) HisTrap HP Column Chromatography

To 12 L of the culture solution of 293F cells expressing humanSiglec-15-His prepared in a) of Example 31, 1350 mL of 10×buffer (500 mMTris, 1.5 M NaCl, 200 mM imidazole, pH 8.0) was added, and the resultingmixture was stirred well and filtered through a MilliPak-60 filter(manufactured by Millipore Co., Ltd.). This culture solution was appliedto a Ni-Sepharose HP (manufactured by Amersham Biosciences, Inc.) 100 mlcolumn which had previously been washed with pure water (Milli-Q water)at a flow rate of 10 ml/min. After the column was washed with 400 ml of50 mM Tris-HCl buffer (pH 8.0) containing 300 mM NaCl at a flow rate of8 mL/min, a protein adsorbed onto the column was eluted with 200 ml of50 mM Tris-HCl buffer (pH 8.0) containing 300 mM NaCl and 500 mMimidazole at a flow rate of 2.5 ml/min, and the eluate was fractionatedinto mini-sorp tubes (manufactured by Nunc, Inc.). In order to preventprecipitation of the protein, 8 ml of a 5 M NaCl solution was added toabout 40 ml of a fraction containing the eluted protein, followed bystirring, and then, the resulting mixture was concentrated to about 20ml with a centrifugal membrane concentrator Amicon Ultra-15(manufactured by Millipore Co., Ltd.). Insoluble matter generated duringthe concentration was removed by centrifugation at 3000 rpm for 30minutes at 4° C., and 2.5 ml of the resulting supernatant was applied toa PD-10 desalting column (manufactured by Amersham Biosciences, Inc.)which was previously equilibrated with phosphate-buffered salinecontaining 1 M NaCl (N-PBS), followed by elution with N-PBS, whereby 3.5ml of a sample whose solvent was replaced with N-PBS was obtained. Thisprocedure was performed 7 more times by repeating it, and about 28 ml ofa solution of partially purified human Siglec-15-His was obtained.

a-ii) Resource Q Column Chromatography

12 ml of the sample which was purified by Ni-Sepharose HP columnchromatography and whose solvent was replaced with N-PBS was dialyzedovernight at 4° C. against 50 mM Tris-HCl buffer (pH 7.5) containing0.1% CHAPS (1 L, three times) and the resulting dialysate wascentrifuged at 3,000 rpm for 30 minutes at 4° C., and the precipitatewas removed. After the resulting supernatant was filtered through aMillex-GV filter (manufactured by Millipore Co., Ltd.), the filtrate wasapplied to a Resource Q 6 ml column (manufactured by AmershamBiosciences, Inc.) which was previously equilibrated with 50 mM Tris-HClbuffer (pH 7.5) containing 0.1% CHAPS at a flow rate of 1 ml/min.Thereafter, the column was washed with this buffer at a flow rate of 1ml/min and a protein fraction which was not adsorbed onto the column wascollected. A protein adsorbed onto the column was eluted with 50 mMTris-HCl buffer (pH 7.5) containing 0.1% CHAPS and 1 M NaCl at a flowrate of 1 ml/min. After 26.5 ml of the fraction which was not adsorbedonto the column was concentrated to 3.0 ml with a centrifugal membraneconcentrator Amicon Ultra-15 (manufactured by Millipore Co., Ltd.), theconcentrate was centrifuged at 3,000 rpm for 10 minutes at 4° C. and theprecipitate was removed. 2.5 ml of the resulting supernatant was appliedto a PD-10 desalting column (manufactured by Amersham Biosciences, Inc.)which had previously been equilibrated with phosphate-buffered salinecontaining 50 mM arginine hydrochloride (pH 7.0, A-PBS), followed byelution with A-PBS, whereby 3.5 ml of a sample whose solvent wasreplaced with A-PBS was obtained. The arginine hydrochloride in thesolvent of the prepared sample was added for preventing soluble humanSiglec-15-His from precipitating. The supernatant after centrifugationwas cryopreserved at −80° C. until use. The above-mentioned purificationprocedure (Resource Q column chromatography) was performed twice byrepeating it.

a-iii) Detection and Purity Assay of Purified Human Siglec-15-his

By using a sample prepared by the above-mentioned purification procedure(Ni-Sepharose HP column chromatography and Resource Q columnchromatography), SDS-polyacrylamide electrophoresis under reducingconditions and silver staining were performed. That is, to 5 μl of eachof the samples purified by the respective purification steps, anequivalent amount of an SDS-treatment solution was added, and theresulting mixture was thermally treated at 95° C. for 10 minutes. 0.3 μlof each of the thermally treated samples was used for SDS-polyacrylamideelectrophoresis. The electrophoresis procedure was performed in the samemanner as the method described in Example 8 except that RainbowMolecular Weight Markers (manufactured by Amersham Biosciences, Inc.)were used as the molecular weight markers. After completion of theelectrophoresis, silver staining was performed using PhastGel Silver Kit(manufactured by Amersham Biosciences, Inc.) and PhastSystem. Theresults are shown in FIG. 28. It was shown that a protein having amolecular weight of about 35 kDa (human Siglec-15-His) was efficientlypurified and concentrated in the protein fraction which was not adsorbedonto the Resource Q column.

a-iv) Measurement of Protein Concentration of Purified HumanSiglec-15-his

For the purified human Siglec-15-His (the protein fraction which was notadsorbed onto the Resource Q column), the protein concentration wasmeasured with a DC-Protein Assay kit (manufactured by Bio-RadLaboratories, Inc.) using bovine serum albumin as a standard sample. Byperforming the purification procedure twice, a total of 1.66 mg ofpurified human Siglec-15-His was obtained.

b) Purification of Soluble Human Siglec-15-Fc

b-i) HiTrap Protein A Column Chromatography

1.5 L of the culture solution of 293F cells expressing humanSiglec-15-Fc prepared in b) of Example 31 was filtered through aSterivex-GV filter (manufactured by Millipore Co., Ltd.), and then, thefiltrate was applied to a HiTrap Protein A 5 ml column (manufactured byAmersham Biosciences, Inc.) which was previously equilibrated withDulbecco's PBS (D-PBS, manufactured by Invitrogen, Inc.) at a flow rateof 5 ml/min. After the column was washed with 70 ml of D-PBS at a flowrate of 5 ml/min, a protein adsorbed onto the column was eluted with 24ml of 0.1 M sodium citrate buffer (pH 3.0) at a flow rate of 1.2 ml/min.The eluate was fractionated at 1.2 ml per fraction into mini-sorp tubes(manufactured by Nunc, Inc.), and immediately thereafter, 0.31 ml of 1 MTris was added thereto to neutralize the eluate. A 2.5 ml aliquot of asolution (about 7.5 ml) obtained by combining the eluted proteinfractions (fractions 5 to 9) was applied to a PD-10 desalting column(manufactured by Amersham Biosciences, Inc.) which had previously beenequilibrated with phosphate-buffered saline containing 50 mM argininehydrochloride (pH 7.0, A-PBS), followed by elution with A-PBS, whereby3.5 ml of a sample whose solvent was replaced with A-PBS was obtained.This procedure was performed twice by repeating it. The argininehydrochloride in the solvent was added to prevent soluble humanSiglec-15-Fc from precipitating. 2.5 ml of the remaining solution of theeluted protein fractions (fractions 5 to 9) was applied to a PD-10desalting column (manufactured by Amersham Biosciences, Inc.) which hadpreviously been equilibrated with phosphate-buffered saline containing 1M NaCl (pH 6.7, N-PBS), followed by elution with N-PBS, whereby 3.5 mlof a sample whose solvent was replaced with N-PBS was obtained. NaCl inthe solvent in the prepared sample was added to prevent soluble humanSiglec-15-Fc from precipitating without adding an amino group-containingcompound such as arginine. The human Siglec-15-Fc sample whose solventwas replaced with N-PBS was used only when an immobilized column wasprepared in the following c) of Example 34, and in all the otherExamples, human Siglec-15-Fc whose solvent was replaced with A-PBS wasused. The samples prepared by the above-mentioned procedure werecryopreserved at −80° C. until use.

b-ii) Detection and Purity Assay of Purified Human Siglec-15-Fc

By using the samples prepared by the above-mentioned purificationprocedure, SDS-polyacrylamide electrophoresis under reducing conditionsand silver staining were performed. That is, to 5 μl of each of thesamples purified by the respective purification steps, an equivalentamount of an SDS-treatment solution was added, and the resulting mixturewas heated at 95° C. for 10 minutes. 0.3 μl of a sample obtained bydiluting each of the thermally treated samples to 1/100 or 1/300 with ahalf concentration of the SDS-treatment solution was used forSDS-polyacrylamide electrophoresis. The electrophoresis and silverstaining were performed in the same manner as the purity assay of humanSiglec-15-His described in a-iii). The results are shown in FIG. 29. Itwas shown that a protein having a molecular weight of about 55 kDa(human Siglec-15-Fc) was efficiently purified and concentrated in theprotein fraction which was eluted from the HiTrap Protein A column.

b-iii) Measurement of Protein Concentration of Purified HumanSiglec-15-Fc

For the purified human Siglec-15-Fc (the protein fraction eluted fromthe PD-10 desalting column), the protein concentration was measured witha DC-Protein Assay kit (manufactured by Bio-Rad Laboratories, Inc.)using bovine serum albumin as a standard sample. As shown in Table 8, atotal of 25.2 mg of purified human Siglec-15-Fc was obtained byperforming the purification procedure twice.

TABLE 8 Protein Conc. Sample (mg/ml) Vol. (ml) Total protein (mg)Arg-containing solvent 2.3 7.0 16.1 NaCl-containing 2.6 3.5 9.1 solventTotal 25.2

Example 33 Production of Rabbit Anti-Human Siglec-15 Polyclonal Antibody(Immunization of Rabbit) a) Preparation of Antigen

The human Siglec-15-Fc protein produced in b) of Example 32 was preparedat 100 μg/0.5 ml, and an equivalent amount of an adjuvant was addedthereto and an emulsion was produced using a glass syringe. As theadjuvant, Freund's complete adjuvant (FCA, Manufactured by DifcoLaboratories, Inc.) was used only for the first immunization, andFreund's incomplete adjuvant (FICA, Manufactured by Difco Laboratories,Inc.) was used for the second and subsequent immunizations.

b) Immunization of Rabbit

Three rabbits (Japanese white female rabbits with a body weight of 3 kg)were used as immunized animals. Incidentally, blood was collected beforeimmunization, and 10 ml of pre-immune serum was obtained per rabbit. Theemulsion obtained in a) was injected subcutaneously and intradermallyusing a 27 G injection needle such that the amount of the antigen was 50μg per rabbit. Immunization was performed a total of 8 times every 14days after the first immunization. The whole blood was collected after 7days from the date of 8th immunization, and 74.4 to 74.9 ml of antiserumwas obtained per rabbit. The antibody titers in the pre-immune serum andthe antiserum were confirmed by an ELISA method using an immobilizedantigen. As a result, an increase in antibody titer in the antiserum wasconfirmed in all the three rabbits. The antiserum was stored at −20° C.until use.

Example 34 Purification of Rabbit Anti-Human Siglec-15 PolyclonalAntibody a) HiTrap Protein A Column Chromatography

To 40 ml of each of the three rabbit antiserum lots prepared in b) ofExample 33, 40 ml of Dulbecco's PBS (D-PBS, manufactured by Invitrogen,Inc.) was added and mixed, and the resulting mixture was filteredthrough a Sterivex-GV filter (manufactured by Millipore Co., Ltd.).Then, the filtrate was applied to a column which comprised two HiTrapProtein A 5 ml columns (the two columns were connected in series,manufactured by Amersham Biosciences, Inc.) and had previously beenequilibrated with D-PBS at a flow rate of 2 ml/min. After the column waswashed with 35 ml of D-PBS at a flow rate of 1 ml/min, a proteinadsorbed onto the column was eluted with 50 ml of 0.1 M sodium citratebuffer (pH 3.0) at a flow rate of 1 ml/min. The eluate was fractionatedat 2.5 ml per fraction into mini-sorp tubes (manufactured by Nunc,Inc.), and immediately thereafter, 0.6 ml of 1 M Tris was added theretoto neutralize the eluate. After about 15.5 ml of a solution obtained bycombining the fractions (fractions 3 to 7) containing the eluted proteinwas concentrated to 5 ml with a centrifugal membrane concentrator AmiconUltra-15 (manufactured by Millipore Co., Ltd.), a 2.5 ml aliquot of theconcentrate was applied to a PD-10 desalting column (manufactured byAmersham Biosciences, Inc.) which had previously been equilibrated withOtsuka Physiological Saline for Injection (TO-SS) containing 0.01% Tween20, followed by elution with TO-SS, whereby 3.5 ml of a sample whosesolvent was replaced with TO-SS was obtained. This procedure wasperformed twice by repeating it. The thus prepared samples werecryopreserved at −80° C. until use.

b) Purification of Pre-Immune Rabbit IgG

Blood had previously been collected from the three rabbits used inExample 33, before initiation of immunization with human Siglec-15-Fc,and pre-immune serum was prepared. After 5 ml aliquots of each of theseserum samples were mixed with one another, 15 ml of D-PBS was addedthereto, and the resulting mixture was filtered through a Millex-GVfilter (manufactured by Millipore Co., Ltd.). Then, the resulting serumsample was applied to a column which comprised two HiTrap Protein A 5 mlcolumns (manufactured by Amersham Biosciences, Inc.) and had previouslybeen equilibrated with D-PBS at a flow rate of 1 ml/min. After thecolumn was washed with 35 ml of D-PBS at a flow rate of 1 ml/min, aprotein adsorbed onto the column was eluted with 50 ml of 0.1 M sodiumcitrate buffer (pH 3.0) at a flow rate of 1 ml/min. The eluate wasfractionated at 2.5 ml per fraction into mini-sorp tubes (manufacturedby Nunc, Inc.), and immediately thereafter, 0.6 ml of 1 M Tris was addedthereto to neutralize the eluate. After a solution obtained by combiningthe fractions (fractions 4 to 6) containing the eluted protein wasconcentrated to 2.5 ml with a centrifugal membrane concentrator AmiconUltra-15 (manufactured by Millipore Co., Ltd.), the concentrate wasapplied to a PD-10 desalting column (manufactured by AmershamBiosciences, Inc.) which had previously been equilibrated with OtsukaPhysiological Saline for Injection (TO-SS) containing 0.01% Tween 20,followed by elution with TO-SS, whereby 3.5 ml of a sample whose solventwas replaced with TO-SS was obtained. The thus purified pre-immunerabbit IgG sample was subjected to polyacrylamide electrophoresis andsilver staining by the method described in Example 8 to confirm that theIgG protein was sufficiently purified, and then, the proteinconcentration was measured. The thus purified pre-immune rabbit IgGsample was cryopreserved at −80° C. until use.

c) Preparation of Affinity Column Having Human Siglec-15-Fc ImmobilizedThereon

3 ml of the purified human Siglec-15-Fc whose solvent was replaced withN-PBS produced in b) of Example 32 (a total of 7.8 mg of protein) wasconcentrated to 2 ml using a centrifugal membrane concentrator AmiconUltra-4 (manufactured by Millipore Co., Ltd.). To the concentrate, acoupling buffer (0.2 M NaHCO₃, 0.5 M NaCl, pH 8.3) was added to bringthe final volume to 2.5 ml, and the solvent was replaced with 3.5 ml ofthe coupling buffer using a PD-10 desalting column. After isopropanol inan NHS-activated HiTrap column (1 ml, manufactured by AmershamBiosciences, Inc.) was replaced with 1 mM hydrochloric acid, 3 ml of theprepared human Siglec-15-Fc was injected into the column using asyringe, and the liquid was alternately injected thereinto using anothersyringe connected to the outlet of the column to effect a couplingreaction. After the reaction was allowed to proceed at room temperaturefor 30 minutes, in order to inactivate excess active groups, 6 ml of ablocking buffer (an ethanolamine buffer containing 0.5 M NaCl, pH 8.3),6 ml of a washing buffer (a sodium acetate buffer containing 0.5 M NaCl,pH 4.0), and 6 ml of the blocking buffer were injected in sequenceaccording to the protocol of Amersham Biosciences, Inc., and then, thecolumn was left at room temperature for 30 minutes. Thereafter, 6 ml ofthe washing buffer, 6 ml of the blocking buffer, and 6 ml of the washingbuffer were injected into the column in sequence again, and finally, thebuffer in the column was replaced with 10 mM Tris-HCl buffer (pH 7.2)containing 0.15 M NaCl. This column was stored at 4° C. until use.

d) Purification of Rabbit Anti-Human Siglec-15 Polyclonal Antibody withAffinity Columnd-i) Affinity Column Chromatography

After 7 ml of each of the purified anti-human Siglec-15 polyclonalantibodies (Nos. 1, 2 and 3) prepared in a) was filtered through aMillex-GV filter (manufactured by Millipore Co., Ltd.), the resultingfiltrate was applied to the column produced in c) which had the humanSiglec-15-Fc immobilized thereon and had previously been equilibratedwith the Apply Buffer at a flow rate of 0.25 ml/min. After the columnwas washed with 5 ml of the Apply Buffer at a flow rate of 0.25 ml/min,a protein adsorbed onto the column was eluted with 5 ml of 0.1 M glycinehydrochloride buffer (pH 2.7) containing 0.5 M NaCl at a flow rate of0.25 ml/min. The chromatograms of the anti-human Siglec-15 polyclonalantibodies (Nos. 1, 2 and 3) purified with the affinity column are shownin FIGS. 30 a-30 c. The eluate was fractionated at 0.5 ml per fractioninto mini-sorp tubes (manufactured by Nunc, Inc.), and immediatelythereafter, 16 μl of 1 M Tris was added thereto to neutralize theeluate. About 2.5 ml of a solution obtained by combining the IgG proteinfractions (fractions 3 to 7) eluted with the glycine hydrochloridebuffer for each antibody was applied to a PD-10 desalting column(manufactured by Amersham Biosciences, Inc.) which had previously beenequilibrated with Dulbecco's phosphate-buffered balanced saline solutioncontaining 0.01% Tween 20 (T-PBS), followed by elution with T-PBS,whereby 3.5 ml of a sample whose solvent was replaced with T-PBS wasobtained. The thus prepared samples were cryopreserved at −80° C. untiluse.

d-ii) Measurement of Protein Concentration of Affinity-Purified RabbitAnti-Human Siglec-15 Polyclonal Antibody

For the purified rabbit anti-human Siglec-15 polyclonal antibody samples(the protein fractions eluted from the PD-10 desalting column), theprotein concentration was measured with a DC-Protein Assay kit(manufactured by Bio-Rad Laboratories, Inc.) using bovine IgG as astandard sample. As shown in Table 9, about 9.1 to 11.9 mg of theaffinity-purified anti-human Siglec-15 polyclonal antibody could beprepared in each of lot Nos. 1 to 3.

TABLE 9 Protein Conc. (mg/ml) Sample Vol. (ml) Total protein (mg) No. 13.0 3.5 10.5 No. 2 2.6 3.5 9.1 No. 3  3.4 3.5 11.9

Example 35 Effect of Addition of Rabbit Anti-Human Siglec-15 PolyclonalAntibody on Cell Fusion of Normal Human Osteoclast Precursor Cells (TRAPStaining)

Normal human osteoclast precursor cells (Normal Human Natural OsteoclastPrecursor Cells, purchased from Sanko Junyaku Co., Ltd., Cat. No.2T-110) were seeded in a 96-well plate at 1×10⁴ cells/well according tothe protocol attached to the cells. As the medium, a minimal essentialmedium for osteoclast precursor cells (OPBM, purchased from SankoJunyaku Co., Ltd., Cat. No. PT-8201) supplemented with an OPGMsupplement set (Osteoclast SingleQuot™ Kit, purchased from Sanko JunyakuCo., Ltd., Cat. No. PT-9501) containing fetal bovine serum (finalconcentration: 10%), human RANKL (final concentration: 69 ng/ml), humanM-CSF (final concentration: 33 ng/ml) and the like was used. To theresulting culture supernatant, the affinity-purified anti-humanSiglec-15 No. 2 antibody produced in d) of Example 34 at a finalconcentration of 3 or 30 μg/ml, or the pre-immune rabbit IgG produced inb) of Example 34 at a final concentration of 30 μg/ml was added, and thecells were cultured for 5 days in a CO₂ incubator. After the culturing,the supernatant was removed, and 10% neutral formalin was added to fixthe cells. After fixing the cells, the cells were washed twice withdistilled water, and a TRAP staining solution (0.27 mM naphthol AS-MXphosphate (manufactured by Sigma Co., Ltd.), 1.6 mM fast red violet LBsalt (manufactured by Sigma Co., Ltd.), 1% dimethylformamide, 50 mMsodium tartrate, 0.1 M sodium acetate buffer (pH 5.0)) was added at 100μl/well, and a reaction was allowed to proceed at room temperature for 5minutes. Then, the cells were washed twice with distilled water, andthen, observed by microscopy (FIG. 31). As a result, the formation ofgiant osteoclasts resulting from a high degree of cell fusion wassignificantly inhibited by the addition of the anti-human Siglec-15polyclonal antibody. On the other hand, in the case where the pre-immuneIgG was added, such inhibition of osteoclast cell fusion was notobserved. The number of TRAP-positive multinucleated cells in which thenumber of nuclei is 5 or more was counted with an inverted microscope(FIG. 32). As a result, a significant inhibition of multinucleatedosteoclast formation was observed in the well to which theaffinity-purified anti-human Siglec-15 No. 2 antibody was added at afinal concentration of 30 μg/ml. Also in the case where the pre-immuneIgG was added at 30 μg/ml, a tendency of inhibition of multinucleatedosteoclast formation was observed. However, when comparison was madewith such wells, it was shown that multinucleated osteoclast formationwas significantly inhibited by the addition of the anti-human Siglec-15No. 2 antibody. In this manner, it was revealed that multinucleation andcell fusion of TRAP-positive osteoclasts from normal human osteoclastprecursor cells are inhibited by the antibody specifically binding toSiglec-15.

Example 36 Evaluation of Binding Property of Rat Anti-Mouse Siglec-15Monoclonal Antibody to Human Siglec-15 Protein

The binding property of the rat anti-mouse Siglec-15 monoclonal antibodyto human Siglec-15 protein was evaluated by an ELISA method. The humanSiglec-15-Fc protein produced in b) of Example 32 was diluted to 5 μg/mlwith 0.1 M sodium carbonate buffer (pH 9.5), and the resulting solutionwas added to a 96-well plate (manufactured by Nalge Nunc International,Inc., Cat. No. 430341) at 100 μl/well. After the plate was left at roomtemperature for 1 hour, the solution was removed and a washing buffer(phosphate-buffered saline containing 0.05% Tween 20) was added at 300μl/well and removed. After this washing procedure was performed one moretime, phosphate-buffered saline containing 25% BlockAce (manufactured byDainippon Sumitomo Pharma Co., Ltd.) was added at 200 μl/well, and theplate was left at room temperature for 1 hour, whereby blocking waseffected. The liquid was removed, and the plate was washed twice with300 μl/well of washing buffer. Then, each of the rat anti-mouseSiglec-15 monoclonal antibodies prepared in Example 24 or rat controlIgG (manufactured by R&D systems, Inc.) was diluted to a finalconcentration of from 1.28 to 20,000 ng/ml (5-fold dilution series) withan ELISA buffer (phosphate-buffered saline containing 12.5% BlockAce and0.05% Tween 20), and the resulting diluted antibody solution was addedto the plate at 100 μl/well. After the plate was left at roomtemperature for 1 hour, the liquid was removed, and the plate was washedthree times with 300 μl/well of washing buffer. Subsequently, HRP(horseradish peroxidase)-labeled goat anti-rat IgG antibody(manufactured by Beckman Coulter, Inc.) diluted to 1,000-fold with theELISA buffer was added at 100 μl/well, and the plate was left at roomtemperature for 1 hour. The liquid was removed and the plate was washedthree times with 300 μl/well of washing buffer, and then, by using acolor developing kit for peroxidase (manufactured by Sumitomo BakeliteCo., Ltd.), the color was developed according to the protocol attachedto the kit. After developing the color, the absorbance at 492 nm wasmeasured using a microplate reader (manufactured by Nihon MolecularDevices Corporation). As a result, it was confirmed that all the 10 testsubstances of the rat anti-mouse Siglec-15 monoclonal antibodiesexamined bind to the human Siglec-15 protein in an antibodyconcentration-dependent manner (FIG. 33). In particular, the bindingactivity of 5 test substances: #1A1, #3A1, #24A1, #32A1, and #61A1, washigh, and the binding activity of 3 test substances: #8A1, #34A1, and#39A1, was low. On the other hand, in the case of the rat control IgG,binding to the human Siglec-15 protein was not observed. From the aboveresults, it was shown that the rat anti-mouse Siglec-15 monoclonalantibodies prepared in Example 24 bind not only to mouse Siglec-15, butalso to human Siglec-15, and moreover, it was found that some antibodiesstrongly bind to human Siglec-15.

Example 37 Effect of Addition of Rat Anti-Mouse Siglec-15 MonoclonalAntibody on Cell Fusion and Bone Resorption Activity of Normal HumanOsteoclast Precursor Cells (Evaluation of In Vitro Biological Activity)

Since it was confirmed that the rat anti-mouse Siglec-15 monoclonalantibodies bind also to human Siglec-15 in Example 36, the effects ofthese antibodies on human osteoclast formation and bone resorptionactivity were examined.

a) Effect of Addition of Rat Anti-Mouse Siglec-15 Monoclonal Antibody onCell Fusion of Osteoclasts from Normal Human Osteoclast Precursor Cells(TRAP Staining)

Normal human osteoclast precursor cells (Normal Human Natural OsteoclastPrecursor Cells, purchased from Sanko Junyaku Co., Ltd., Cat. No.2T-110) were seeded in a 96-well plate at 1×10⁴ cells/well according tothe protocol attached to the cells. As the medium, a minimal essentialmedium for osteoclast precursor cells (OPBM, purchased from SankoJunyaku Co., Ltd., Cat. No. PT-8201) supplemented with an OPGMsupplement set (Osteoclast SingleQuot™ Kit, purchased from Sanko JunyakuCo., Ltd., Cat. No. PT-9501) containing fetal bovine serum (finalconcentration: 10%), human RANKL (final concentration: 66 ng/ml), humanM-CSF (final concentration: 33 ng/ml) and the like was used. To theresulting culture supernatant, each of the rat anti-mouse Siglec-15monoclonal antibodies prepared in Example 24 or rat control IgG(manufactured by R&D systems) was added to give a final concentration of30 μg/ml, and the cells were cultured for 4 days in a CO₂ incubator.After the culturing, the supernatant was removed, and 10% neutralformalin was added to fix the cells. After fixing the cells, the cellswere washed twice with distilled water, and a TRAP staining solution(0.27 mM naphthol AS-MX phosphate (manufactured by Sigma Co., Ltd.), 1.6mM fast red violet LB salt (manufactured by Sigma Co., Ltd.), 1%dimethylformamide, 50 mM sodium tartrate, 0.1 M sodium acetate buffer(pH 5.0)) was added at 100 μl/well, and a reaction was allowed toproceed at room temperature for 5 minutes. Then, the cells were washedtwice with distilled water, and then, observed by microscopy (FIG. 34).As a result, the formation of giant osteoclasts resulting from a highdegree of cell fusion was almost completely inhibited by the addition ofthe #32A1 antibody. Further, also in the case of the #41B1 antibody, theformation of giant osteoclasts resulting from a high degree of cellfusion was significantly inhibited. On the other hand, in the case ofthe other rat anti-mouse Siglec-15 monoclonal antibodies (such as the#1A1 antibody) and the rat control IgG, such a significant inhibition ofosteoclast cell fusion was not observed. In this manner, it was revealedthat multinucleation and cell fusion of TRAP-positive osteoclasts fromnormal human osteoclast precursor cells are inhibited by the monoclonalantibody specifically binding to the Siglec-15 protein.

b) Effect of Addition of Rat Anti-Mouse Siglec-15 Monoclonal Antibody(#32A1) on Cell Fusion of Osteoclasts from Normal Human OsteoclastPrecursor Cells (TRAP Staining)

Normal human osteoclast precursor cells (Normal Human Natural Osteoclast

Precursor Cells, purchased from Sanko Junyaku Co., Ltd., Cat. No.2T-110) were seeded in a 96-well plate at 1×10⁴ cells/well according tothe protocol attached to the cells. As the medium, a minimal essentialmedium for osteoclast precursor cells (OPBM, purchased from SankoJunyaku Co., Ltd., Cat. No. PT-8201) supplemented with an OPGMsupplement set (Osteoclast SingleQuot™ Kit, purchased from Sanko JunyakuCo., Ltd., Cat. No. PT-9501) containing fetal bovine serum (finalconcentration: 10%), human RANKL (final concentration: 68.4 ng/ml),human M-CSF (final concentration: 33 ng/ml) and the like was used. Tothe resulting culture supernatant, the rat anti-mouse Siglec-15monoclonal antibody (#32A1) prepared in Example 24 was added to give afinal concentration of 0.1, 0.3, 1, or 3 μg/ml, and the cells werecultured for 3 days in a CO₂ incubator. After the culturing, thesupernatant was removed, and 10% neutral formalin was added to fix thecells. After fixing the cells, the cells were washed twice withdistilled water, and a TRAP staining solution (0.27 mM naphthol AS-MXphosphate (manufactured by Sigma Co., Ltd.), 1.6 mM fast red violet LBsalt (manufactured by Sigma Co., Ltd.), 1% dimethylformamide, 50 mMsodium tartrate, 0.1 M sodium acetate buffer (pH 5.0)) was added at 100μl/well, and a reaction was allowed to proceed at room temperature for 5minutes. Then, the cells were washed twice with distilled water, andthen, observed by microscopy (FIG. 35). As a result, the formation ofTRAP-positive multinucleated osteoclasts was inhibited in a #32A1antibody concentration-dependent manner within the range of from 0.3μg/ml to 3 μg/ml.

c) Effect of Addition of Rat Anti-Mouse Siglec-15 Monoclonal Antibody(#32A1) on Bone Resorption Activity of Normal Human Osteoclast PrecursorCells (Evaluation Using Collagen-Coated Plate)

It is known that osteoclasts release a protease such as cathepsin K anddegrade type I collagen which is a constitutional component of bonetissue. OsteoLyse Assay Kit (manufactured by Lonza, Inc., Cat. No.PA-1500) provides a 96-well plate coated with europium-conjugated humancollagen (96-well OsteoLyse cell culture plate), and it is possible toevaluate the bone resorption activity of osteoclasts in vitro bymeasuring the amount of fluorescent collagen fragments released in thesupernatant when osteoclasts are cultured in the plate.

Normal human osteoclast precursor cells (Normal Human Natural OsteoclastPrecursor Cells, purchased from Sanko Junyaku Co., Ltd., Cat. No.2T-110) were seeded in a 96-well OsteoLyse cell culture plate at 1×10⁴cells/well according to the protocol attached to the cells. As themedium, a minimal essential medium for osteoclast precursor cells (OPBM,purchased from Sanko Junyaku Co., Ltd., Cat. No. PT-8201) supplementedwith an OPGM supplement set (Osteoclast SingleQuot™ Kit, purchased fromSanko Junyaku Co., Ltd., Cat. No. PT-9501) containing fetal bovine serum(final concentration: 10%), human RANKL (final concentration: 68.4ng/ml), human M-CSF (final concentration: 33 ng/ml) and the like wasused. To the resulting culture supernatant, the rat anti-mouse Siglec-15monoclonal antibody (#32A1 antibody) prepared in Example 24 was added togive a final concentration of 0.1, 0.3, 1, or 3 μg/ml, and the cellswere cultured for 3 days in a CO₂ incubator. A 10 μl aliquot of theculture supernatant was collected, and 200 μl of Fluorophore ReleasingReagent included in the OsteoLyse Assay Kit was added thereto, and afluorescence intensity was measured (Excitation: 340 nm, Emission: 615nm) using a fluorescence plate reader (ARVO MX, manufactured by PerkinElmer Inc.), whereby the amount of free fluorescent collagen fragmentsreleased in the culture supernatant was determined (FIG. 36). As aresult, the amount of fluorescent collagen fragments increased by theaddition of RANKL was reduced by the #32A1 antibody in aconcentration-dependent manner within the range of from 0.3 μg/ml to 3μg/ml. From this result, it was revealed that the bone resorptionactivity of human osteoclasts is inhibited by the monoclonal antibodyspecifically binding to the Siglec-15 protein.

Example 38 Production of Mouse Anti-Human Siglec-15 Monoclonal Antibody

It is possible to produce an anti-human Siglec-15 antibody by the stepsdescribed below.

(1) Immunization

The soluble human Siglec-15 protein obtained in Examples 30 to 32 isintraperitoneally administered to a female BALB/c mouse at the age of 4to 10 weeks. After 2 weeks, the same membrane fraction solution isintraperitoneally administered to the mouse for booster immunization.

(2) Cell Fusion

The spleen is resected from the mouse on three days after the boosterimmunization, placed in a serum-free medium and crushed on a mesh with aspatula. The cell suspension passed through the mesh is centrifuged toprecipitate the spleen cells.

On the other hand, myeloma cells NS1 (American Type Culture CollectionTIB-18) are washed with a serum-free medium and suspended in the samemanner.

By using the thus obtained Siglec-15-expressing cells and myeloma cells,cell fusion is performed according to a common procedure.

(3) Screening

Anti-human Siglec-15 antibody-producing fused cells can be screened by amethod using cell-ELISA and a method using a flow cytometer.

(4) Cloning

For a group of cells screened in the above (3), a series of stepscomprising the method using cell-ELISA and the method using a flowcytometer described in (3) is repeated 5 times, whereby several clonesof hybrdomas capable of producing a single antibody which binds to humanSiglec-15-expressing cells, but does not bind to the cells beforetransfection can be obtained.

(5) Purification of Antibody

A mouse-mouse hybridoma produced through steps (1) to (4) is cultured,and the resulting supernatant is collected. After the obtainedsupernatant is collected and dialyzed, partial purification of theantibody is performed using a high performance liquid chromatographyapparatus. The anti-human Siglec-15 antibody titer in each fraction ofchromatography is assayed by an ELISA method using the human Siglec-15protein. Fractions having a high antibody titer are collected andapplied to an antibody affinity purification column. After the inside ofthe column is washed with a column equilibration buffer, the antibody iseluted with a column elution buffer. Immediately after completion of theelution, each eluate is applied to the top of a centrifugal ultrafilterand centrifuged. After the filtrate collected in the bottom of thefilter is removed, washing is performed 5 times by adding PBS to thetop. The liquid remaining on the top of the filter is used as ananti-human Siglec-15 antibody sample.

Example 39 Production of Rat Anti-Human Siglec-15 Monoclonal Antibody

A rat anti-human Siglec-15 monoclonal antibody can be produced using thesoluble human Siglec-15 protein obtained in Examples 30 to 32 by themethod described in Examples 23 and 24.

Example 40 Effect of Anti-Human Siglec-15 Monoclonal Antibody onOsteoclast Differentiation

By using the anti-human Siglec-15 monoclonal antibody obtained inExample 38 or 39, the inhibitory effect of the antibody on osteoclastformation can be tested. For testing the effect on mouse osteoclastformation, the method of Example 17, 19, 20, 21, 22 or 26 can be used.For testing the inhibitory effect on human osteoclast formation, themethod of Example 35 or 37 can be used.

INDUSTRIAL APPLICABILITY

The anti-Siglec-15 antibody of the invention has the ability to inhibitosteoclast differentiation or bone resorption activity, and apharmaceutical composition containing the anti-Siglec-15 antibody can bea therapeutic or preventive agent for a disease of abnormal bonemetabolism.

1. An antibody or an antigen binding fragment thereof, whichspecifically recognizes any one of the following polypeptides (a)-(i),or any combination thereof, and inhibits osteoclast formation and/orosteoclastic bone resorption: (a) the amino acid sequence of SEQ ID NO:2; (b) amino acid residues 21 to 328 of the amino acid sequence of SEQID NO: 2; (c) amino acid residues 1 to 260 of the amino acid sequence ofSEQ ID NO: 2; (d) amino acid residues 21 to 260 of the amino acidsequence of SEQ ID NO: 2; (e) the amino acid sequence of SEQ ID NO: 4;(f) amino acid residues 21 to 341 of the amino acid sequence of SEQ IDNO: 4; (g) amino acid residues 1 to 258 of the amino acid sequence ofSEQ ID NO: 4; (h) amino acid residues 21 to 258 of the amino acidsequence of SEQ ID NO: 4; or (i) an amino acid sequence includingsubstitution, deletion, or addition of one or more a min acid residue inthe amino acid sequence described in (a) to (h) wherein the amino acidsequence has a biological activity comparable to that of the amino acidsequence described in (a) to (h).
 2. The antibody or antigen bindingfragment of claim 1, wherein the antibody is a monoclonal antibody. 3.The antibody or antigen binding fragment of claim 1, wherein theantibody is an IgG antibody.
 4. The antibody or antigen binding fragmentof claim 1, wherein the antibody or antigen binding fragment competeswith an antibody produced by hybridoma #32A1 (FERM BP-10999).
 5. Theantibody or antigen binding fragment of claim 1, wherein the antibody orantigen binding fragment has the same epitope specificity as an antibodyproduced by hybridoma #32A1 (FERM BP-10999).
 6. The antibody or antigenbinding fragment of claim 1, wherein the antibody or antigen bindingfragment competes with an antibody produced by hybridoma #41B1 (FERMBP-11000).
 7. The antibody or antigen binding fragment of claim 1,wherein the antibody or antigen binding fragment has the same epitopespecificity as an antibody produced by hybridoma #41B1 (FERM BP-11000).8. A pharmaceutical composition comprising an antibody or antigenbinding fragment according to claim 1, wherein the pharmaceuticalcomposition further comprises a pharmaceutically acceptable diluent orcarrier.
 9. The pharmaceutical composition of claim 8, wherein thepharmaceutical composition further comprises at least one agent selectedfrom the group consisting of bisphosphonate, active vitamin D₃,calcitonin, hormones, ipriflavone, vitamin K₂ (menatetrenone), calcium,PTH (parathyroid hormone), nonsteroidal anti-inflammatory agent, solubleTNF receptor, anti-TNF-α antibody or an antigen binding fragmentthereof, anti-PTHrP (parathyroid hormone-related protein) antibody or anantigen binding fragment thereof, anti-IL-6 receptor antibody or anantigen binding fragment thereof, anti-RANKL antibody or an antigenbinding fragment thereof, and OCIF (osteoclastogenesis inhibitoryfactor).